Biophysical Chemistry Upadhyay

BIOPHYSICALCHEMISTRY PRINCIPLES AND TECHNIQUES BIOPHYSICAL CHE vIISTRY PRINCIPLES AND TECHNIQUES AVINASH UPADHYAY, M.Sc., Ph.D. Department of Biochemistry, Hislop College, Nagpur (M.S.) KAKOLI UPADHYAY, M.Sc., Ph.D. Department of Biochemistry, Lady Amritabal Dga College, Shankarnagar, Nagpur (M.S.) " NIRMALENDU NATH, M.Sc., Ph.D. Retired Professor, Department of Blchemistr, Nagpur University, LIT Premises, Nagpur (M.S.) %Iimalaya ¢PublishingHouse MUMBAI DELHI 0 NAGPUR BANGALORE HYDERABAD Nag u r Bangalore © AUTHORS No part ofthis book shall be reproduced, reprinted or translated for any purpose whatsoever withou prior permission of the publisher in writing. First Edition : 1993 Second Revised Edit/on : 1997 Reprint : lkprkt : 2000 Rprlnt : 00. eprint : Third Revised Edition : 2002 (May) Repr/nt : 2003 Published by : Mrs. Meena Pandcy for HIMALAYA PUBLISHING HOUi, "Ramdoot, Dr.Bhalerao Marg, Girgaon, Mumbai - 400 004. Phones : 2386 01 70/2386 38 63 Fax : 022 - 2387 71 78 F.-mail : himpub @ vsl.com Website: www.h/mpub,©om Branch Offices : Delhi : Hyderabad "Pooja Apartments", 4-B, Murari Lal Street, Ansari Raod, Daryaganj, New Delhi - 110 002. Phone : 2327 03 92 Fax : 011 - 2325 62 86 : Kundanlal Chandak Industrial Estate, Ghat Road, Nagpur - 440 018. Phones : 272 12 15 / 272 12 16 : No.16/1, (Old 12/1) First Floor, Next to Hotel Highland, Madhava Nagar, Race course Road, Bangalore - 560 001. Phones : 228 15 41,238 54 61, Fax : 080-228 66 11. : No.2-2-I 167/2H, 1st Floor, Near Railway Bridge, Tilak Nagar, - Main Road, Hyderabad - 500 044. Phone : 5550 17 45, Fax: 040-2756 00 41 : Geetanjali Press Pvt. Ltd., Kundanlal Chandak Industrial Estate, Ghat Road, Nagpur - 440 018. PROF. CHANDRA NTH PIONEER OF IDIAN BIOCHEM/STRY PREFACE TO THE THIRD REVISED EDITION One of the problems facing the writers of textbooks in fast moving subjects is t he obsolescence of old information and sprouting of new knowledge. Luckily, for us, even with tremendous growth in biochemistry, the basic techniques of study have remai ned unaltered. Yet, variations and improvi.ations of the old techniques have been pe rfected since the book was last revised. The present resion is an attempt to take into a ccount these changes. The Achilles' heel of the previous edition was the chapter on centrifugation. Th is was brought to our notice time and again by students and teachers of the subject from diverse corners of the nation. We are happy to state that the chapter has been t horoughly revised, almost rewritten, and the scope of the discussion has been greatly expa nded. Another aspect that has been completely revised is infrared spectrometry. Again it was brought to our eyes by people who have taken critical notice of the book. Th e current discussion of the aspect is again much wider in its scope and care has been take n to give several examples of its use in biochemistry and allied sciences. New techniques such as fluorescence energy transfer, fluorescence polarization, non-radioactive labeling, etc. have been added. Also, radioimmunoassay has been discussed more extensively along with a good discussion of its variant, radioimmunometry. A sore point with the previous editions was the lack of anindex. This shortcom, g has been addressed and the book now has a detailed index. The above are the major additions. Numerous small changes in virtually all the will become visible to the teachers of the subject who have seen the first two editions. New problems have also been added at the end of the chapters. Late in the day, when we had already submitted the revised draft for publication , we received the UGC syllabi for Biochemistry, Microbiology, Botany, Zoology, Biotec.hnology, and otherallied life science subjects We were pleasantly surpris ed to draftnot only covered all major points but had more to offer leaving in the syllabi. The. current edition will be of good use to students any life science subject. In its revised form, we feel confident that the book win be much more useful to all concerned. We would like to thank the great number of teachers and students who have praise d our book; they provided us the support that eyery author nxis so much. We are eq ually to those who criticized the book for providing the motivation to revise it. ACKNOWLEDGEMENTS A book of this expanse does not bcome possible without contribution of several willing souls. We have been lucky that several colleagues and students helped us in our endeavour in whichever way they could, sometimes even going out of the way to do it. We would like to thank Prof. H. F. Daginawala, former Head of the Department of Biochemistry, Nagpur University, for his constant encouragement and help. We are also highly indebted to Dr. N. V. Shastri, Head of the Department of Biochemistry, Nagpur University, for his suggestions regarding the changes to be made in the revised edition. The fact that he taught this very subject to two of the authors (AU & KU) has !.everything to do with the writing of the textbook. . Heartfelt thanks are due to Ms. Ragi Radhakrishnan, one of our ,cherished students, for designing the cover for the 3rd edition and for going through much of the 2rid edition in search of mistakes, typographical otherwise. Dr. Rajnish Kaushik and Dr. Shibani Mitra Kaushik, St. Louis, Missouri, out of the way to provide us as much current literature as they could of the fact that both of them were laden with their own research work. cannot really express our feeling of gratitude towards them. We are indebted to Dr. Saraswati Sukumar (Johns Hop.kins), and Dr. Runnebaum, The Salk Institute, La Jolla, USA, who provided us of much of the recent literature. Thanks are also due to several of our colleagues, friends and students Dr. Irfan Rahman, Dr. Ashish Bhelwa, Dr. Saibal Biswas, Dr. S.N. Ms. Sadhana Naidu, Ms, Ramfla Bhojwani, Dr. Raymond Andrew, Dr. Deshpande, Mr. Amol Amin, Mr. S. Wankhede, Dr. Shyam Biswal, Ms. Anjali Gadkari. We record our appreciation of Mrs. Harsha Dave who was involved in ,g the Ist edition. Finally, we would like to thank Shri Gokul Pandey and Shri D. P. of Himalaya Publishing House for their constant help and valuable 1 - 65 66- 74 75 - 99 CONTENTS ACIDS AND BASES Electrolytic Dissociation and Electrolytes -- Ionization: Basis of Acidity and Basicity w Bronsted-Lowry Theory: Acid is a Proton Donor, Base is a Proton Acceptor -- Strength of Acids and Bases -Acid-Base Equilibria in Water -- Function and Structure of Biomolecules is pH Dependent w Measurement of pH: Use of Indicators m Electrometric Determination of pH -- Buffers: Systems which Resist Changes in pH -- Titrations: The Interaction of an Acid with a Base. ION SPECIFIC ELECTRODES Ion Selective Electrodes Measure the Activity of Metal Long m Glass Membrane Electrodes m Solid-State Ion Exchanger Electrodes Solid-State Crystal E lectrodes Liquid-Membrane Electrodes w Gas-Sensing Electrodes. 3. THE COLLOIDAL PHENOMENA Classification of Colloids -- Properties of Colloids -- Donnan Equilibrium. . . DIFFUSION AND OSMOSIS A Molecular-Kinetic approach to Diffusion m Methods of Determination of Diffusion Coefficient Significance of Diffusion Coefficient w D iffusion of Electrolytes -- Diffusion of Water Across Membranes: Osmosis Measurement of Osmotic Pressure Van't HoiTs Laws of Osmotic P ressure --Theories of Osmotic Pressure and Semipermeability m Osmotic Behaviour of Cells m Molecular Weight Determination from Osmotic Pressure Measurements -Significance of Osmosis in Biology. VISCOSITY Factors Affecting Viscosity Measurement of Viscosity Applications of Viscornetry Significance of Viscosity in Biological Systems. I00- 121 122- 144 SURFACE TENSION Factors Affecting Surface Tension w Measurement of Surface Tension. 145-156 ADSORFtlON Kinds of Adsorption Interactions -- Adsorption Characteristics -Molecular Orientation Adsorption Isotherms: Quantitative 157- 174 Relationships -- Adsorption from Solutions n The Importance of Adsorption Phenomena. 8. SPECTROPHOTOMETRY Basic Principles -- The Laws of Absorption n Significance of Extinction Coefficient (Box) Problems (Box) -- Preparation of Standard Graph (Bo x) Deviations From Beer's Law -- Absorption Spectrum -- Why is Absorption Spectr um Specific For A Substance? -- The Chromophore Concept -- Instrumentation For UV-Visible And Infrared Sprectrophotometry -- Radiant Energy Sources -Wavelength Selectors -- Detection Devices -- Amplification And Readout Double Beam Operation -- Double wavelength Spectrophotometer Application s of UV-Visible Spectrophotometry Qualitative Aalysis -- How to Interpret Absorp tion Spectra of Biological Macromolecules (Box) -- Quantitative Analysis Enzyme Assay n Molecular Weight Determination -- Study of Cis-trans Isomerism Other Physicochemical Studies -- Control of Purification Difference Sp ectrophotometry Turbidimetry and Nephelometry -- Theory and Applications of Infr ared Spectrophotometry -- Calculation ofVibrational Frequencies -Modes of Vibration Infrared Spectra of Common Functional Groups -- The Carbon Sk eleton -- Carbonyl Group -- Hydroxy Compounds Nitrogen Compounds -- Infrared Spectrophotometer: Mode of Operation Sa mpling Techniques -- Applications of Infrared Spectrophotometry -- Disadvantages of Infrared Spectrophotometry -- Spectro.fluorimetry Structural Factors Which give Rise to Fluorescence -- Fluorescence and Phosphorescence (Box) -- Fluorometry: Theory and Instrumentation -- Applications -Fluorescence Spectra and Study of Protein Structure -- Extrinsic Fluorescence -- Fluorescence Energy Transfer -- Fluorescence Polarization n Luminometry -- Flame Spectrophotometry -Instrumentation for Emission Flame Photometry Instrumentation for Atomic Absorpt ion Spectrophotometry -- Atomic Fluorescence -- Nuclear Magnetic Resonance Spectrophotometry -- Magnetic Properties of the Nucleus -- Nuclear Resonance Chemical Shifts: Position of Sign als '-- Hyperfine Splitting -- Instrumentation Applications Electron Spin Resona nce Spectrometry n Applications -- Spin Labeling -- Mossbauer Spectrophotometry -Applications Some Solved Problems 9. OTHER OPTICAL TECHNIQUES FOR MOLEC CHARACTERIZATION Circular Dlchrolsm and Optical Rotatory Dispersion -- Rotational Diffusion -- Flow Bircfringence D E1ectrlc Birefringence D Polarization of Fluorescence -- Light Scatterlng-- X-ray Diffraction. 175-270 271-300 12. 10. CENTRIFUGATION Basic Principles of Centrlfugatlon m Relative Centrifugal Force (RCF) -- Other Factors Affecting Sedimentation -- Instrumentation -Desktop Centrifuge -- High Speed.Centrifuge The Ultracentrifuge -- Analytical Ultracentrifuge Fixed-angle Rotors -- Vertical-tube rotors Swingin g-bucket Rotors n Wall Effects Preparative Centrifugation -- Differential Centri fugation -- Density Gradient Centrifugation -- Rate Zonal Centrifugation -- Isopycnic Centrifugation Gradient Materials Preparation of Density Gradients --Choice of R otors Centrifugation in Zonal Rotors --Centrifugation Analytical Basic Principle s of Centrifugation --Factors Affecting Sedimentation Velocity -- Sedimentation Coefficient -- Factors Affecting Standard Sedimentation Coefficient Measurement of Sedimenta tion Coefficient Concentration Distribution -- Applications Of Boundary Sediment ation -- Band Sedimentation Determination of Molecular Weights 301-343 II. CHROMATOGRAPHY 344-421 Survey of Chromatographic Procedures -- Techniques of Chromatography -- i. Plane Chromatography -- A. Paper Chromatography B. Thin-Layer Chromatography 2. Column Chromatography --Types of Chromatography -- 1. Chromatography 2. Partition Chromatography A. Liquid-Liquid Chromatography B. Gas-Liquid Chromatog raphy (GLC} -- 3. Gel Permeation Chromatography 4. Ion Exchange Chromatography 5. Affinity Chromatography High Performance Liquid Chromatography Some Specialized Technique s -- Hydroxyapatite Chromatography -- An Affinity System for Base Dependent Fractionation of DNA -An Affinity System for Fractionating supercoiled and NonSupercoiled DNA -- DNA-Cellulose Chromatography. ELECTROPHORESIS 422-478 Migration of an Ion in an Electric Field m Factors Affecting Electrophoretic Mobility -- Types of Electrophoresis 1. Free Electrophoresis 2. Zone Electrophoresls. General Techniques of Zone Electrophoresis -- 1. Paper Electrophoresis 2. Cellulose Acetate Electrophoresis 3. Gel Electrophoresis. Specialized Electrophoretic Techniques I. Discontinuous (Disc) Gel Electrophoresis 2. Gradient Electrophores is 3. High Voltage Electrophoresis (H.V.E.) 4. Isoelectric Focussing 5. Two-Dimensional Gel Electrophoresis 6. Immunoelectrophoresis 7. Pulse-Field Gel Electrophoresis 8. Electrophoresis on Cellular Gels. Electrophoresis in Genetic Analysis 1. Restriction Mapping. 2. Southern Transfer. 3. Gel Retardation or Ban d Shift Assay. 4. DNA Sequencing. 5. DNA Foot printLng. - 14. 13. ISOTOPES IN BIOLOGY ,Radioactive Decay =- Production of Isotopes -- Synthesis of Labeled Compounds -- Interaction of Radioactivity with Matter m Measurement of Radioactivity -- 1. Methods Based Upon Gas Ionization --A. Ionization Chambers B. Proportional Counters C. Fundamentals of Geiger Counters 2. Photographic Methods 3. Methods Based Upon Excitation w A. Liquid Scintillation Counting Use of Stable Isotopes in Biology -- The TracerTechnique -- Use of Isotopes as Tracers in Biological Sciences -- Some Information About Commonly Used Isotopes -- Safety Aspects -Dosimetry. CERTAIN PHYSICOCHEMICAL TECHNIQUES USEFUL IN BIOCHEMISTRY Polymerase Chain Reaction -- Enzyme-Linked Immunosorbent Assay (ELISA) -- Flow Cytometry. 479 - 54, 546 - 56! -- APPENDICES -- INDEX 567593 - 60: 1 ACIDS AND BASES A history of the quest to understand the molecular basis of acid - base properti es ,m.akes for a very amusing reading. For instance, in 1773 Doctor Samuel Jhonson averred that acids iare composed of pointed particles which affect the taste in a sharp and piercin g manner". iAnother attempt to explain the nature of acids was made by Lavoisier when he pr oposed that i the characteristic behaviour of acids was due to the presence of oxygen. Stimula ted by this observation, Sir Humphrey Davy went to great lengths to show that hydrochloric a cid also contalns oxygen. He, of course, failed in his attempt thereby disproving the the ory of Lavoisier. Even the later history of acid - base research is not without its share of amuse ment, albeit in :a manner different to the above described instances. In 1884 Svante August Arrh enius in his dissertation proposed the theory of electrolytic dissociation and ionization on which current understanding of acid - base character is based. The doctoral dissertati on was, greeted by the lowest possible pass-mark by the University of Uppsala, Sweden. F or , Arrhenius was awarded Nobel Prize in Chemistry in 1903. ELECTROLYTIC DISSOCIATION AND ELECTROLYTES Let us consider a simple experiment. A pair 1.1. F.Jcperlmental system for determining electrical conductivity of a solutWn. The bulb does not light when there is a nonbulb l@hts when the beaker cohtalns eteces n of electrodes is connected in series to a light bulb and to a source of electricity (Figure 1.1). As long as the electrodes hang separated in the air, no electric current flows through the circuit, and the bulb does not light. If however, the two electrodes are touched to each other, the circuit is completed and the bulb lights. If the electrodes are dipped into a beaker containing water purified by repeated distillations, the bulb does not light. This tella us that water is not a good conductor of electricity and is not capable of completing the circuit. If we dissolve an acid, a base, or a salt in water in which the electrodes are dipped, the bulb lights up. Obviously, these substances are able to carry the current and thereby complete the circuit. Substances produc/ng solutions capable of conductlng electric'.tty are called electrolytes. On the other hand, substances producin9 solutions incapable of conducting electricity are known as non-electrolytes. Table 1.1 provides a few examples of electrolytes and non-electrolytes. 2 Bophs. Chemis What is the mechanism by which, electrolytes conduct electricity? Arrhenius' the ory proving an answer. The theory proposes that acids, bases, and salts undergo dissociation in water varying degrees, each molecule giving rise to oppositely charged long. For examp le, if gase¢ hydrogen chloride is bubbled into water, virtually all the hydrogen chloride mol ecules re. with .water (Figure1.2) giving rise to a hydronium ion (positively charged) and a chloride i {negatively charged}. These long can now be carried to the cathode and the anode respectiv thereby completing the circuit. This theory of Arrhenius is known as the theory of electrol3 dissociation. Water Hydrogen Chloride Collision 'Complex' Hydronium Ion Chloride Io: Figure 1.2. When gaseous hydrogen chloride is bubbled in water, HCI mo/ecu/es co llide with water molecu Collisions of sufficient energy and proper orientation produce hydronlum long an d chloride long. Going back to the experiment we discussed, a diligent observer would note that c erta substances cause the bulb to be brightly lit, whereas other substances cause the bulb to only dimly lit. This experimental observation permits us to subdivide the electr olytes into groups. Substances that dissociate almost completely and produce sol utions that are very go conductors of electricity are known as strong electrolytes; substances which dis sociate only part and produce solutions which are poor conductors of electricity are known as weak electroly The difference between strong and weak electrolytes was attributed by him to a d ifference in t degree of ionization. IONIZATION : BASIS OF ACIDITY AND BASIClTY Arrhenius Theory : H÷ Ion is the Acid, OH- Ion is the bae Fom the experiment that we have discussed above, one can safely conclude that ac i base reactions are a function of ionization p-nciple. Thus, based on ionization princip nhenius defined acids and bases. These definitions are elaborated below. Acls : Acids were described by Arrhenlus as compounds containing hydrogen will upon addition to water become ionized to yield 14+ long. Nitric acid (14N03), wh ich is a solut strong electrolyte or srong ac [Le,, it dissociates completely in water to produ ce t4+ long), m be cited as an example. HNO3 H÷ + NO Nitrous acid (HNO2) , a weak electrolyte {Le., dissociates only partially to pro duce H+ iont may be cited as an example of a weak acid. HNO2 # H+ +NO. (A single arrow ----> denotes reactions that go completely to the right; a double arrow x--- denot re.actions that go only partially to the right). 3 Acids and Bases Table 1.1 Examples of Electrolytes and Nonelectrolytes Strong Electrolytes Hydrochloric acid, HCI [H+ + Cl-] . Nitric acid, HNOa [H+ + NO] Sulfuric acid, H2SO4 [H+ + HSO] Sodium hydroxide, NaOH [Na+ + OH-] Potassium chloride, KCI [K+ + CI-] Silver nitrate, AgNO3, [Ag+ + NO ] Sodium chloride, NaCI [Na÷ + CI-] Copper fib sulphate, CuSO4 [Cu2+ + SO-] Weak Electrolytes Nonelectrolytes Acetic acid CH3COOH [CHaCOOH] Lactic acid, CHaCHOHCOOH [CHaCHOHCOOH] Ammonia, NH3 [NHa] Hydrogen sulphide, H2S [H2S] Mercury (II} chloride, HgC12 [HgCI2 ] Glucose C6H1206 [C6H1206 ] Sucrose C12H22011 [C12H22011 ] Ethyl alcohol, C2HsOH [C2H5OH ] Methyl alcohol, CH3OH [CH3OH ] Acetone CH3COCH3 [CHaCOCHa ] Species in parentheses are predominant in solution. The difference between weak and nonelectrolyte is that weak electrolytes dissociate very lltfle (not shown in th e table) whereas the nonelectrolytes do not dissociate at all. Bases : According to the Arrhenius definition, bases are compounds which upon io nization in water yield OH- (hydroxide) long. Sodium hydroxide, which dissociates complet ely to produce OH- long, may be cited as an example. NaOH Na+ + OHThe Arrhenius concept is important in that it has provided us with the first mec hanistic approach to acid - base behaviour and has been instrumental for the development of more sophisticated theories. There are, however, two major shortcomings in the Arrhen ius model. "- (0 In the AIThenius model the acid-base reactions are limited to aqueous solu tions (this is not a problem as far as biological systems are concerned since all reactions must take place in aqueous solutions). (//) The theory limits bases to hydroxide compounds. This is very unsatisfact ory because it is well known that many organic compounds which are not hydroxides, for examp le ammonia, show basic properties in their chemistry. In the year 1923, two more theories defining acid-base character were proposed. The first theory, Bronsted and Lowry theory, is very satisfactory for understanding physio logical processes and will therefore form the basis of all further discussions. The second theory, proposed by G. N. Lewis is much more general than the Bronsted - Lowry concept. A brief disc ussion of this theory is given in Box 1.1. 4 Biophysical Chemistr Bronsted - Lowry Theory :' Acid is a Proton Donor, Base is a Proton Acceptor This theory defines an acid as any compound that yields protons (H+ long) and a base as any compound that combines with a proton. In other words, acids are proton do nors and bases are proton acceptors. It should be noted that as-far as acids arc concerne d, Arrhenius and Bronsted - Lowry theories are similar ; in both cases acids give off H+ long . However, the concept of a base is much broader in the Bronsted theory, hydroxyl ion being jus t one of the possible bases. Cited below are a few examples which will illustrate the point m uch better. general equation H2SO4 H+ HSO HC1 H+ " + C1HsPO4 H+ [ + HuPO CHaCOOH H+[ + CHaCOOHCOa H+ [ + HCO HCO H+ I + CO - HsO+ H+{ + H.O HA H+ I + A- Concept of conjugate ac/d and conjugate base : Each of the compounds listed abov e as acid, pon ionization, produces. H+ long. Their ionization also produces long or molecu les which can ombine with a proton (HSO , Cl-, H2PO , CHsCOO-, etc). According to the definiti on, these which can combine with a proton are bases: Thus, we can say that every acid diss ociates to a proton and a base (if the reaction is reversed, a base can combine with a proton to an acid}. The Bronsted -Lowry theory thus conceives of an acid base 'pair'. An acid its corresponding base are said to be 'conjugate', i.e., 'joined in a pair'. Thu s, CI- is the of HCI, likewise H20 is the conjugate base of H30+. An acid is a proton donor. Its strength would depend upon the ease with which it can a proton. An acid will yield a proton with comparative ease if its conjugate bas e is weak. HCI as an example. Its conjugate base, CI-, is a weak base; it is not a very goo d In solutions, therefore, HCI is completely ionized to produce H÷ and CI-. HCI IS i strong acid because/ts conjugate base/s wea/ Let us consider another example, that of Its conjugate base CH COO- is stronger base compared to CI-. The acetate ion, binds the proton much more tenaciously with the result that in solution acetic a cid is ' ionized. CHCOOH Is a weak ac/d because/ts conjugate base Is strong. Similar co ncepts e drawn for bases also and their strength would epend upon the strength of their conjugate The Bronsted Lowry theory gives us the following reciprocal relations : -- ff an acid is strong, its conjugate base is weak: -- if an acid is weak, its conjugate base is strong. -- if a base is strong, its conjugate acid is weak. if a base is weak, its conjugate acid is strong. Concept of an a/ka/i : In the previous pages NaOH was regarded as an Arrhenius b ase ionized to produce OH- long. NaOH, however, is not a Bronsted base because, as a it has little ability to accept a proton. NaOH can act as a base solely because upon it gives rise to OH- long which are very good proton acceptors. NaOH and other hydroxides like KOH, therefore act as bases by proxy. Such compounds, under the theory, are known as alkalies. the dis cussic applies equally well to bases. in a reverse manner). Thus. under the Bronsted concept. Both these factors are discusse . However. however. liquid ammonia qual ifies as acid NH3 NH + H+ and as a base too -. is not the only determinant of st rength. In a preceding section we have said that the strength of an acid depends upon th e strength weakness of its conjugate base. CH3CO0 CH3CO0 Acid Ionizable Salt Metal Hydrogen STRENGTH OF ACIDS AND BASES (Throughout the dlscussiori. NH3 + H+ NH Similar is the case with water which behaves as an acid HOH x. and (ii) the dielectric constant of the solvent. This. Apm from strength of conjugate base. albeit. Thu CH3COONa is the sodium salt of CH-COOH formed by replacement of the proton by th e N ion.6 Biophysical Chemisl Amphoteric substances : Substances which can behave both as an acid and as a bas e referred to as amphoteric.I-IO* Sa/ts : Under this tleory salts are thought to be compounds which are formed by replaci the ionizable hydrogen with a metal ion or with any other positively charged gro up.H÷ + OHand as a base HOH + H+ --. KCI is a salt of HCI formed by replacement of the proton by K+ ion. the strength of an acid depends upon (i) the ba sic strength ¢ the solvent. acids will be treated as examples. as a solvent. that of water. th e H÷ long {formec due to ionization of an acid) are known to combine with water molecules to give rise to H30+ the.below. H++H20 ---.--x H+ + and the general ionization reaction of acids as HA --. The Basic Strength of the Solvent So far we have been writing the ionization reaction of HCI as HCI .H++A It is. well known that H+ long do not exist in acid solutions. Let u s illustrate th caseby considering a specific example.H30+ . hydronlum long (also known as the oxonium or hydroxonium ionsl. This is because th H+ long combine with the solvent molecules to give rise to 'lyonlum long'. however. In water. is a weak acid in water. A= Mill 10se ionlzable hydrogen to water Zhich is a stronger base. In th-is cae A.Lowry concept states that a base is a proton acceptor./f the basic strength of the solven t is less base. Case 2 : A. Therefore. The dissociation of the ac id. 0 O CH3-C-OH+H-OH CH--C--O:+H30+ " Case 2 : Acetic acid in liquid ammonia. the ackl will be strong in that solvent. and H20 to accept the Ionlzable hydrogen. The acetate ion is a stronger base than water. Acetate ion is a weaker base as compa red to Therefore. HA. now is a function of the competition between the tw o bases. acetic acid which was a weak acid in water.' The strerth of the acid. HA. will be less and it not be a stror acld in water. We can now rewrite the general lonition reaction of an acid In water HA+H20H30+÷A.. A a consequence. In this case. Coe I : A= is strorer than H20. HA. Case I : Acetic acid in water. let us consider the strength of the same acid in two so lvents.is weaker than H-O. once the acid is dissolved in wate r. is a strong acid in liqui d O O . The acid.iAds m Boes 7 Recall that Bronsted . Th us water the above cae (and solvents in general} is acting as a base. the dissociation of acld.. Mill and the acid may even be completely dissociated.eak in that solvent. To drive the point home. If the basic strength greater tbn that of the conjugate base. . We can now generalize the above observations. HA. the ac will be v. will be a strong in water.Is a stronger base and bind to t he bI hydrogen much more tenaciously than H20. in a solvent which has a low dielectric constant. This action o f the acid and consequently is important for the strength of acid. Constant of the Solvent Upon ionization the acid splits into two oppositely charged long. However. The se long attract each other and recombine. H+ and A-. It can thus be said that the ran acid s always relative to. will not dissoc iate much . The that an acid is strong does not convey much sense unless we know in relation to directlon of proton transfer and its extent depend upon these relative proton donating proton-bindlng abilities of the potential acids and the solvent.the basic strength of the solvent used. solvents of high dielectric constant greatly attraction between oppositely charged partlcles dissolved in them. acid in a solvent of high dielectric constant Mill dissociate greatly and will t herefore be The same acid.CH3-C-O-H+NH3 CH3-C-O-+NH The above examples show the relative nature of the designations strong and weak. Carboxylic acids which contain strong electron at tracting grou (halogens) on the alpha .2.carbon are stronger than the unsubstituted acids. therefore.+ H+ 8 B/ophysicat Chemts and will consequently be weak. Effectof Structure on the Strength of -cids It is a commonly accepted fact that carboxylic acids are stronger than other org anic aci Why is that so? The reason usually given is that the carboxylate anion (the conj ugate ba formed upon dissociation is stabilized by resonance (two equivalent resonance st ructures] such a manner that it is more stable than the original acid molecule. almost 80. A given acid can therefore dissociate to a much greater exte nt in water tt in petroleum ether. On t he other ha carboxylic acids bearing electron releasing groups (methyl) on the alpha . the negative charge is not delocali zed an( concentrated on the single oxygen atom. in the alkoxide ion. This anion. is not as stable as the resonar stabilized carboxylate anion. If resonance stabilization were the only factor all carboxylic acids would have had t same strength.ngth of an acld.Water is a solvent which has a very high dielectri c eonstan' room temperature. The dielectric constant is thus of great importance in deter mining stre. petroleum ether has a very low d ielee constant.carbo . The resonance stabilization promotes dissociation in the carboxy acids making them stronger in relation to the organic acids where lack of resona nce stabfllzati decreases dissociation. This is not so. TO-. On the other hand. o l R--C// u" + R--C \oH Resonance stabilized anion On the other hand. just 2. C Cl<---.n atom a weaker than the unsubstituted acids. C1 0 CI O (I) CI¢-----C<---.H CI O + . Electron attracting groups withdraw electrons from the carbox ylate grou This weakens the oxygen .C <-----.hydrogen bond thereby facilitating ionization and rele ase of a proto Moreover. These electrostatic factors.C C1 O <--. in which elect rons are eith attracted to or repelled from one atom or group of atoms with respect to another are known inductive effects. these groups also help stabilization of the conjugate base by resonanc e. If.Acids and Bases 9 Inductive effects are additive and increase with the number of substitutions by electron withdrawing or electron releasing groups. in true sense. When we said th at CH. It may be said that the activity and concentration of H÷ long might be identical in dilute solutions. substitution by a halogen on the beta carbon of a carboxylic acid is not as effe ctive as one on the alpha . Thus. Thus.COOH is a weak acid. however. The concept ofactW/ty : The long in soluUon. are separated from one another by s hielding layers of solvent and thus have little attraction for each other. however. The long then assume a certain degree of orientation. It is only in the concentrated solutions that they start to differ. This effective concentration is k nown bya better term. Thus. the strength of an acid is a measure of the activity of H÷ long and not of its concentration. the long can not m ove freely because they are closer to each other and therefore are affected by oppositely c harged long. the intervening distances between different long start decreasing. Each ion is surrounded by an ' ion atmosphere' of opposite charge which reduces its movement. We. however. Thus the effective concentration of the long is sllghfly less than its absolute concentration. what we meant was that HCI ionizes t¢ give a high [H÷]. w e increase the concentration of the solution. These effects also are sensitive to di stance. the long move about freely without the hindrance of attrac tive forces from oppositely charged long. we meant that CH3COOH ionizes to only a little extent giving a l ow [H+].carbon. activity. In a concentrated solution. Activity coejc/ent : Activity is a measure of the effective concentration of the solutes in The activities might be related to the absolute concentrations by a proportional . AIIhough this is the way we are going to use this term subsequently in this chapter. have been using t he term loosely to convey in essence the H÷ ion concentration [H+] . What Do We Mean by 'Strength of an Acid'? So far we have not reviewed this term critically. we m ight as well understand its actual meaning. when we said th at HCI is a strong acid. In a dilute solution. The symbol used for activity coefficient is ¥. HCI al so conducts current much better than CHsCOOH.01 N NaOH are required to fully tltrate 25 ml of 0. The two acids. Acetic acid is wea kly 1. have similar titration 25 ml of 0. The r coefficient approaches unity at infinite dilution. however. the long liberated from it combine with the H÷ present and remove them in the form of water.ity called activity coefficient. Both acids give titration response because of the following scheme of events. The eq uation for the relationship is as follows a=¥C a is the activity and C the concentration. HCI ion izes 'and almost all the hydrogen of HCl is present as H+ at any point of tim.3% of its hydrogen is present as H+. Units of both a and C are moles per l itre.+ H+ NaOH Na÷ + OHH20 .01 N HCI.0 IN CH3COOH. CHsCOOH --. When we add alkali to this solution.CH3CO0. of 0. Does Not Reflect The Strength of an Acid We know that HCI is a considerably stronger acid as compared to CH-COOH.0 IN NaOH is required to fully titrate 25 m. The same of 0. A similar process takes place with HCI also ' HC1 C1. ACID-BASE EQUILIBRIA IN WATER The free hydronium ion concentration. Since all physiological fluids are aqueous based.3 % H*. [H3 O÷]' doininates chemical reactions In ph ysiologic systems. . The acidity me asured titration is known as the total or the titratable acidity and reflects th e concentration of an a in solution. To understand the complex equilibria which are always pr esent in acid . living and t vitro physiological systems. For example. These too are removed as water in the manner d escribed abov The process continues till all acetic acid has ionized to give up protons which get removed = water. Adjusting and controlling the free hydronium ion concentration is a necessity in an biochemical experiment.base system is therefore of paramount importance. It does not. the rate at which it go es.+ H+ + . C and D. NaOH -------> Na÷ + OHHO The two acids therefore end up giving similar titration profiles. According to the law of mass action the rate of the reac tion to the righ will depend upon the molar concentrations of A and B (throughout the discussion we assum that the solution is dilute and thus activity is equal to concentration) Thus I 0 Bophyslcal Chemls To achieve equilibrium with respect to dissociation.in which two reactants A and B interact to form two products. specific enzyme activity is often quite dependent o n the effectiv concentration of the hydronium ion. or tt detailed mechanism of how it takes place in such solutions. reflect the strength of an acid or its actual acidity. however. the concentration of hydronium for may determine the extent to which the reaction proceeds. in both . Note that th reaction is reversible. more acetic acid molecules dissocia to give rise to another 1. In the above equation. . therefore. evolved mainly by Guldberg and Waage. as one would recall. The chemical affinities are con stant at a give: temperature and other reaction conditions. the active mass means the effective conc entration or t13 activity (which might be equal to molar concentration in dilute solutions). for long. and V is t h Apart from the molecular concentration. IBI where [A] and [B] are expressions of molar concentrations of A and B.The Law of Mass Action The law of mass action. The active mass for molecules is essentially equal to their molar concentration However. Let us consider the reaction A+Bx--C+D Vr a [Al. r the reactants should also be taken into account. states that the ra te of chemical reaction at a given time is proportional to the active masses of reacti ng substances prese at that time. the'chemical afllfities ¢ reaction velocity to the right. we might introduc a proportionality constant which corrects for the particular chemical affinity. The acidity me asured b] titration is known as the total or the titratable acidity and reflects the conce ntration of an acia in solution. more acetic acid molecules dissociatl to give rise to another 1. It does not. For example.3 % H+. The I. states that the ra te of a .UILRIA WATER The free hydronium ion concentration.Bi .10 Bophgsical Chemistry To achieve equilibrium with respqqt to dissociation.. specific enzyme activity is often quite dependent on the effective concentration of the hydronium ion. living and h vitro physiological systclns. Adjusting and controlling the free hydronlum ion concentration is a necessity in any biochemical experiment. in both .tion The law of mass action. reflect the strength of an acid or its actual acidity.+ Na+ OH- H* NaOH + HeO The two acids therefore end up giving similar titration profiles. To understand the complex equilibria which are always pr esent in an acid . evolved mainly by Guldberg and Waage. A stmflar process takes place with HC] also 'HC1 C1. however. [H30÷]..base system is therefore of paramount importance. the concent ration of hydronium long may determine the extent to which the reaction proceeds. dominates chemical reactions in phys iological systems. ACID-I-. the rate at which it go es. or the detailed mechanism of how it takes place in such solutions. These too are removed as water in the manner d escribed above The process continues till all acetic acid has ionized to give up protons which get removed a water.w of Ma . Since all physiological fluids are aqueous based. we might introduce a proportionality constant which corrects for the particular chemical affinity. C and D.chemical reaclon at a 9hen time s proportlonoJ to the active masses of reactj su bstances present at that time. the active mass means the effective conc entration or the activity (which might be equal to molar concentration in dilute solutions). thechemi cal aiIties of the reactants should also be taken into account. as one would recall. Let us consider the reaction A+B-C+D in which two reactants A and B interact to form two products. The active mass for molecules is essentially equal to their molar concentrations. According to the law of mass action the rate of the reac tion to the right will depend upon the molar concentrations of A and B (throughout the discussion we assume that the solution is dilute and thus activity is equal to concentration} Thus V a [A]. In the above equation. The chemical affinities are con stant at a given temperature and other reaction conditions. However. for long. therefore. and V is t he reaction velocity to the right. Note that the reaction is reversible. . Apart from the molecular concentration. [BI r where [A] and [B] are expressions of molar concentrations of A and B. the products C and D will react to give A and B.. of Water As per the collision theory. [BI = K2 [Cl. So that and / [A]. it is expected that water molecules constantly coll ide with water molecules. It may further be expected that at any instant a minute fractio n collisions might give rise to the following change: .. when Kis small. IB] Since the reaction is reversible. Writing expression for the velocity of the reaction to the leR V! = K2 It). [D] .. the rate of the reaction to the right and that to the left will be equal. which means that affinity between A and B is higher than that at equilibrium the concentration of C and D is higher than that of A and B.tB] K.AddsandBases 11 Thus vr = K1 IA). The equilibrium equation may be stated in words : at equilibrium the product of the of the substances formed in a chemical reaction divided by the product of of the reactants in that reaction is a constant referred to as the equilibrium /v We may stress again that the activities of the reacting species will give the and not the molar concentrations which are used only for the sake of simplicity.is the equilibrium constant and is an expression of the chemical affinit ies of obvious from the above equilibrium equation that if K is large the the right predominates. [D] [C]. = where . The law of chemical equilibrium may be applied to virtually all reversible react ions and including the ionization of acids and bases.. At equilibrium..[D] KI [A]. This reaction is better illustrated in Figure 1.3. . However. the produc of ionization. This means that water is almost a nonelectrolyte. These experiments also tell us that at e quilibrium a small percentage of water molecules becomes ionized. per lit-re in pure water is equal to the number of grams of H20 in 1 L d ivided by the gra . The equilibrium constant of such a reactic according to the law of mass action would be -] = We have seen that water has a very slight tendency to ionize.O÷. This means that t! . The Equilibrium Constant and Ionization Constant of Water We have seen that water has only a very slight tendency to ionize. But the water will stay essentially neutral because an equa lly higher numb of hydroxyl long will also form.Biophysical Chemistry Figure 1. ion is also produced. we can think that hydronium ion is the hydrated form of H÷ and take t he liber to express tas H÷ for the sake of simplicity). The dissociation of water has b een confirme by electrical conductivity experiments. T he concentration of wat. It is obvious from the above equation that for every single hydronium ion formed . At higher temperatures the number collisions between water molecules will be higher producing a sllghtly higher nu mber hydronium long.. It is therefo re necessary th we express the extent of ionization of water quantitatively. We can represent the ionization of water simply as H--OH H++OH(although we have said before that H÷ long do not exist as such and the correct re presentatic would be H. actually Just slightly more tha 10-7%. concentration of water should be virtually unchanged by ionization.have very profound biological effects. Thus ionization of water forms these two long in equal num bers therek ensuring that pure water is essentially neutral. A collision between two water molecules can result n the formation o f a hydronlwn ion and a hydrox ion.3. H30+ and OH.clent energy and proper orlentation . The collision should be of su. a hydros. molecular weight. 1000/18 = 55. Le.5 2 M or 0.55 x 10 M. Substituting this value in equilibrium constant expression we get . 9 x 10-18 = [H+] [OH-] 1. I × I0-14-.[I-gl [OH-] I X 10-14-.found to be 1.5) (1. 55. basic or neutral .8. and concentration of water. we get [H+I = I x 10-7 .-x 10-16 at 25°C.0 x I0=14 = [H÷I [OH-] at 25"C. In olutions the product of hydrogen ion concentration and hydroxyl ion concentratio n is constant value 10-I. the solution is said to be neutraL. 1. When concentrations of [H+] and [ OH-] as in pure water. we get [H+l [OH-] '. /¢ = 1.8 x 10-16 55.2. of the value of Kw allows us to calculate the concentration of H+ and OH-.5.atlon constant or the dssocfation cons tant or ion product of water and is symbolically denoted as K Thus.13 [H+] [OH-] From electrical conductivity measurements of water thevalue of Keq has been calc ulated il very carefully and has been.8 x 10-Ie = [H*] [OH-] 99.[H+]2 solving for H+. is known as ion7. The above equation is substantially true for water and for dilute aqueous soluti ons. Under such conditions. is taken to be constant.5 " Rean'angg (55. Value of Kw widely with temperature as shown in Table 1. Substituting this value for K in the above equation.4 (at 25"C) whether the solution is acidic.0 x I0-14 ffi [H*] [OH-] The product of the equilibrium constant of water He. 8 x 10-14 24 1.[H+]= [OH-] = 1 x 10-TM Ionization Constant of Water at Var/ous Temperatures Temperature ('C} Ionization Constant (K I0 2.0 × 10-14 25 1.92 x 10-s 18 0. On ther hand.13 x 10-14 Thus when a solution is neutral.47 × 10-14 37 (Normal body temperature) 3.2 X I0-14 30 1. ff the solution is acidic. the concentrations of H÷ and OH.are both 10-7 M. the concentration of H+ would be higher th an 10-7 and . 01 Nfor NaOH at 24=C. Thus a solutio n which is 0. Ans.I 4 Bphysmd Chem that of OI-I.would be less than 10-7 M. Therefore [OH-] of the so lution is 0. The [OH-] of this solution will be .01 Nwith respectto OH-.would be higher th an Thus. = 1 x 10-4 we get [H+]. Ans. (1) Calculate the [H+] of the solution which is 0.001 N for HCI at 24'C. the concen of H÷ should be less than 10-7 M while the concentration of OH. The following examples demonstrate it.001 Nwith repect to H+. when the solution is basic . Therefore. Putting this value into the equati on [H*] [OH"] = K=.01 g tool per litre or 1 x 10" g tool per litre. [H+]of the solution is 0.I X 10-= = I x 10"4 Therefore 0-14 IH* = = 1 x 1 Og tool per Iltre 1 X 1 12 lx10-= (2) Calculate the [OH'] of the solution which is 0. the ionization constant of water. Kw. HCI is a strong acid and by definition dissociates fully respect to HCI is also 0. Furthermore. and vice versa. NaOH is a strong alkali and by definition dissociates fully.01 N with respect to NaOH is also 0.001 litre or I x 10"a g tool per lltre. The relationship [H*] [OH-] = Kw = 1 x 10"4 helps in ¢elcution of [H*] if[OH-] is known and Vke versa. is of great help to calculate the co ncentration ff the concentration of OH.is known. mole/litre. scientist routinely have to make hundreds and thousands of measurements.I OH.Concentrations: The Concept of pH Because of the importance of trace concentrations of hydrogen and hydroxyl long. (ii) 2. (/) [H+] = 1 x 10"e mole/litre. (iv) [H+] = 2 x 10-2mole/litre. (i) lx10-s mole/litre. (ill) 4x 10-e Simple Way of Denoting H+ and OH. .= lx10" lx10'a =1x10"11 g rnol per litre (3) Calculate the [OH-] for each of the following neutral. Arts. (ii) [H+] = 4 x i0-e mole/litm. (iii) [H7 = 2. The manipulation of sucl . The temperature is 24'C.5 x 10"mole/litre.5x10-s (iv) 5 x 10-13 mole litre. M 0 1 2 3 4 . This shortcut is thepH scale which is a co nvenient tool to designate the actual concentration of H÷ (and therefore of OH-) in any aqueous solution in t he range of acidity between 1.3 The pH Scale pH [H*].g.000000 I) cumbersome and tedious.0 MH÷ and 1.8). 10-7) or even their decimal equivale nts (0. The pH of this solution would be given by I pH = log-lx10-7 = log (1 x 107) = log 1. and conversely. the scale rang es from to 14 (Table 1. the chemists. As a matter of simple convenience. 1. the term pH is def'med by t he equation Let us see how convenient is the pH scale.0 M OH-.and Bases 15 wkward figures as negative exponents (e.. Mathematically. is into the simple pH value of 7.0 + log 107 =0+7 pH= 7 Thus the cumbersome figure of neutrality. Solutions which are acidic will have pH values le ss 7. M pOH [OH-]. chiefly Sorensen. ng ago devised a shortcut. ion product of water (1 x 10-). the solutions which are alkaline will have pH values larger t han 7. hydrogen ion concentration of 10-7 M. We know that the hydrogen ion concent ration n a neutral solution at 25° is 1× 10-7 M. forms the basis for the pH scale. 0 I0-l 10-2 10-3 io-4 10-5 10-6 10-7 10-8 10-9 i0-0 i0-I 10-12 10-13 i0-14 14 13 12 Ii i0 .5 6 7 8 9 10 11 12 13 14 1. 0) has . Thus.0 It is necessary to understand that the pH scale is logarithmic and not arithmati c.9 8 7 6 5 4 3 2 l 0 I0-4 10-13 10-12 10-t 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3 102 i0-I 1. Thus. it ineans that o ne solution I0 times the hydrogen ion concdntration of the other. is said that two solutions differ from each other by 1 pH unit. vinegar (pH 3. Table 1.4).000 times greater than that of blood (pH 7. .H÷ approximately 10.4 lists values of some important and commonly used aqueous fluids. 4 Place of Various Materials in the pH Scale Biophysical Chem¢ Mater/al pH value Household bleach Household ammonia Baking soda Sea Water Egg white Hepatic duct bile Intestinal Juice Pancreatic Juice Blood (human} Tears (human) Cerebrospinal fluid Saliva Urine Milk Kupffer cells (intracellular) Black coffee Beer Tomato Juice (ripe) Orange juice Vinegar Cola Lemon Juice Pure gastric juice .16 Table 1. 45 7.0 2.log IOH-] = .log Kw = 14 but. It is important to note that the pH scale is applicable accurately only to solut .concentration) of a giv solution.4. and .5 6.3 2.6 6.4.3 3.8 6.log [H÷] = pH.0 7.6 . slmflarly .0 7.4 7.0 0.4 8.85 7.0 6.0 8.0 8.0 3.5 8.354.5 7.0 9.2 .12.5 4.9 6.log [OH-] = pOH.5 7.9 If we take the negative logarithm of the equation Kw = [H+] JOWl = 10-4 we get log IH÷I .9 4.4 6.0 8.log Kw = pKw Thus pH + pOH = pKw (at 24"C) Sometimes the expression pOH is used to denote basicity (OH.4 5.7 12. .357. ions ordinm-y temperature (approximately 24"C) where the value for pKw is 14. Moreover. measu rement ofpH blood and urine can give us important diagnostic information. Only at this temperatu pH of neutral solutions will be 7. Measurement of pH is of utmost importance to biologists in general and to bioche mists particular. . This is so since pH determines not only the activity of blomolecules such as enzyme but may also be important for the stability of their structures. phenol red. The law of mass action can be applied to formulate equilibrium equations for the dissociation If weak acids are given the genera/formula HA. Weak acids (and weak bases) occur commonly in biologlca l are responsible for metabolic regulation. Measurement of pH of an aqueous solution can be-performed by using such indicato r dyes Isee later) as phenolphthalein. their dissociation equation can as .It must be emphasized that the actual meaning of pH is the negative log of hydro gen ion r and not the hydrogen ion concentration. activity is essentially equal to the concentration. However. Accuratemeasurements. of Weak Acids A biologist is more concerned with the behaviour of weak acids which are not com pletely when dissolved in water. etc. require specially glass electrodes which are very accurate (see later). in dilute solutions with whic h we [ deal. h owever. litmus. I 0 is much more iod aeflc acid wch has a vue of I.86 4.87 . ese vues c be better hdl if e conveed to e negative log us.74 x 10-3 1.9 x 10-7 5. A discussed eer in the secon on pH.35 x 10-s 7.14 3. lactic acid with a Ka of I. s aut0mafl¢ prodes e oaon at lacc acid is a songer acid as comped to acec acid.86 a s comped to 4. Acid pKa Phosphoric acid (H3PO4) Formic acid (HCOOH) Lactic acid (CHaCHOHCOOH) Acetic acid (CH^COOH) Propionic acid CHaCH2COOH) Carbonic acid (H2CO3] Ammonium ion (NH ) 7. dlssociaon constt us deles e tendency of y acid.62 x 10-1° 2.5 ts e d pv of some coon we acids. Table 1.25 x 10-3 1.38 x 10-4 1. A hler vue obviously means higher degree of ionization. us. it is cumbersome to hdle negave eo vues. it should be remembered at s vue would be less stronger ad d m ore for a weber acid. og = p fle mng use of p.18 Bophystcal Chem According to the law of mass action the equilibrium expression for this dissocia tion be written as " where Ka is the dsn cot or the nan t ofwe acid. .75 3. 74 x I 0-s. Thus.76 4. to lose its proton. lactic acid wch is songer acetic has a p vue of 3.78 x 10-4 1.76 of acetic acid. the re certain polybasic acids. like H2C03. We will take H2CO3 as an ex ample. involvi dissociation constant for each stae.1 9. H3PO4 etc..31 x 10-11 where K and K are the first and the second dissociation constant of the acid. Th e dissociat i 2 of the first hydrogen ion from H2CO3 IS opposed by the force of attraction of it s linkage to I .6. whose dissociation should also be conside Polybasic acids dissociate in stages and an equilibrium expression for each stag e.=K2-. However. ' dissociation of H2CO3 takes place as follows: H2CO3 H÷ + HCO 2HCO xH+ + CO 3 We can write equilibrium expressions for each of the two stages -.6. may be written.25 We have so far considered the dissociation of monobasic acids only. [!ydrogen ion concentration so calculated will be approximate as we have not co rrected it for slight dissociation which the weak acid has undergone. One can therefore assume that the concentrati on of the imdissociated acid. The value of K2 is therefore l ess than KI. For the dissociation of a weak acid HA HA : H+ + A flae equilibrium expression may be written as H+ A-] le encentrations of H÷ A.would be equal as they are formed In equal amounts. . molecule.Rgds and Bases 19. v. Thus. The dissociation of the second proton is more difficult. The dissociation constant of a weak acid may be employed to calculate the pH of its 01ution of a known concentration. This value can th en be substituted the above equation and the hydrogen ion concentration can be calculated.erefore. The procedure for such calculation is enumerat ed below. we can safely assume th at not !note than 1% exists as H+ and A-. is equal to the normality of the acid. HA. It is held no t only by the primary union with the molecule but also by the attraction of the negative charg e left on the molecule by the dissociation of the first proton. [H+ 12 = Ka [HA] or [H+ ] = -/-a [HA] We know that weak acids are only slightly ionized. The val ue of . It iS therefore nonsense to write equflibrlum equations and equilibri. the activit y of strong ac might be less than the hydrogen ion concentration produced by ionization.20 1 Biolhysal Chemis Ionization of Strong Acids Strong acids (and bases) dissociate completely or almost completely in dilute aq ue solutions (HCI. cannot i any possible way provide either hydrogen long or hydroxyl long in water.1 N solution of HCI is essentially 0. There are numerous salts which. many of these salts test either acld/c or basic: Thus sodium. from an Inspect. a 0. Th pH of str acids of a given concentration is therefore slightly differerit than what is pre dicted by th concentration. the higher number of long p roduced by strong acids promotes interactior between these long and ttlerefore. constants for their dissociation. Thus. On the other hand. H2S04). CHsCOONa. Yet.. Hydro13is of lts .ion of their chemical formula e. acetate. in solution. test s basic when-ll .1 N in hy drogen coneentration also. . Because NaOH is a strong base (Ikali) . . we will see that this ion is derived from a weak ac id. CH3COO-.O* and OH.concentration. produced by sodium acetate. relydissolved in water. can.ve already seen that weak acids are weak because their conjugate bases are strong and bind th protons rather tenaciously reducing the extent of dissociation.e only way sodium acetate can test basic is by decreasing the HsO+ concentration thus relatively increasing the oH. does not lie in Na+ long. ion in this cse. into the Na+ !ons and acetate.. and Bases 21 . NH4CI. the acetic acid. and by definition ium long readily relgase OH. a H3O÷ long are being produced in such salt solutions? H' To . Apa from these two S. Inspection of the formulae of these salts tells us that th ey can jt podkibly provide H30* or OH" ions. If now we nsider CH3COO. Let us find out whetle r any of the two COO" and Na÷. This inspection us that t. tests acidic in water which means that this solution has 3°÷ long than itlhas OH-.answer this qqestion let us find out which long will be present i n a solution of these lalts tn water.long th an it has H30 + . have the ability to combine with protons effectively reduce their concentration. First let us consider sodium acetate: Upon dissolution i n water. thus. therefore bind with protons (H3O+) and effectively .long. Can Na+ long bind H30+ ? Obvious ly no.or tr.What then is the mechanism by which extra OH.long. The s trong conjugate base. long. Can * long combine with OH.will also be present through dissociation of water. We . some H.4.1. long ? No. sodium acetate mpletely dissociates. Obviously sodium acetate solution has more OH. The answer. ammonium chloride. Sa/ts o f weak acids bases test basic when dissolved in water. The olution #:Lcomes alkal{ne. Th depletes long of wat er wh{le OH" rerna unchared. . however.long. CHo CO0-. Na÷ and OH" do n ot have a tendency to combffe because they are derWed from strong alkali.remove them solution leaving an excess of OH.O Long from the sal : Na÷ CH3CwO- Long from water: H÷ OH' Tenden'y to combine No posslbility of Interaction Hydrolysis of sodgun acetate (salt of strong base and weak acid).4). Is i that all salts of weak acids and strong bases (sodium acetate is an example) basic in soltion? Yes. We therefore can generalize the situation. This makes a so/llum acetate sol ution test basic 1. by definition all of them have strong conjugate b ases which can protons iom solution. has a tendency to combine w[tn H* of water becaus [t s a base covate to a weak ad. (HO÷ shown as H'for the sake of stl ctty) . The solution becomes acidic Figure 1. none of the salt lons has any tenc to combine with either l-I* or OH-.5}. This depletes OH. OHH÷ OH- Figure 1.weak bases test acidic when dissolved in water. hydro: is said to occur.5. As evldent fror figure. The solution re neutral. Hydrolysis of sodium chloride (salt of s acid and strong base).6. We can generalize these situations also and state that (0 sa lts of strong c and.pro by tonlzatton of water. Hydrolysis of ammonium chloride (salt of strong acid and weak base).22 Biophysico2 Cherr The same train of logic can be adapted to find out what will happen when salts of st acid and weak base {Figure 1. Long from salt : NH CILong from salt : Na÷ CI- Long from water Long from water .6} dissolved in water. . If any ion from the salt interacts with water in such a manner as to change its pH.tons of water while-14+ tons remain constant. Cl. NH on the other hand can combine with OH.long. and (il) salts of strong aci ds and strong I test neutral when dissolved in water. and salts of strong acid and strong base {Figur e 1.and I-I* have no tendency to combine. is very strong and binds protons tenaciously. the acetate ion. the H÷ ion concentration gets reduced and the increases. /. Thus.. We can therefore say that the pH of a solution of weak acid and its s alt is determ -by the ratio of salt to acid in the solution. This obviously m eans the addition of salt to acid solution has decreased the dissociation of the acid . Experimental results with such mixed solutions tell us that the pH of such solui increases as compared to the pH when only the acid was present.e. by adding this salt we are further increasing the concen tration o! conjugate base. If we appl) same principles which we considered in the above section.The Effect of Salts Upon the Dissociation of Acids Let us see what happens when a salt of a weak acid is mixed with the weak acl solution. whereas acetic acid is only weakly dissociated. sodium ace as an example. The higher the salt concentration . therefore. CH3COOH CH3COO. From our discussion above we know that sodium acetate is complete ly dissoci in solution. Since sod ium ace dissociates completely. have the follo dissociation equations : .6 elaborates the effect of changing salt to acid ratio on the pH of salt -acid solution . CH3COO'.+ Na+ We have seen that acetic acid is a weak acid and dissociates very little. We.+ H+ CH3COONa ---) CH3COO. the higher the Table 1. Let us consider the solution of acetic acid and its sa lt. we can provide an answ er for decrease in dissociation. These extra long then combine with the sm all numb protons dissociated from acetic acid. This i s becaus conjugate base. 6 4.2 0.05 0.2 0.2 0.7 4.25 0.00 0. where the diss .23 tdds and Bases sble 1.4 4. We might cite the example of NH OH and NH4CI.7 The same set of principles discussed above apply in the case of solutions of wea k hydroxides d their salts also.50 ' 0.0 2.20 0.6 Effect ofChanging Salt/Acid Ratio on the pH of Salt-Acid Solution Sodium Acetate Acetic Acid Rio pH (Molar) (Normal) Salt/Acid 0.75 1.10 0.2 0.2 0.6 4.15 0. OH thereby decrea sing OHcentration and thus a drop in pH restflts. The higher the sal concentration the lower the BUFFERS : SYSTEMS WHICH RESIST CHANGES IN pH Solutions which contain both weak acids and their salts are known as buffer solu tions Ooy same logic. combine with the strong conjugate base are thus removed. NHCI dissociates completely. The pH of the solution does not decrease appreciably (it fails to the change in ratio of salt to acid in solution). acetat e long. From the discussion in the previous know that sodium acetate dissociates fully and acetic acid dissociates only a li ttle. However.ociations 4 NH4OH NH + OHNH4CI --> NH + ClUe NH4OH is only partially dissociated.+ H+ + CI. The could have decreased the pH. Let us now see what happens when an acid or a base is added to thi s When an acid (HCI) is added : CH3COO.]he principle behind this resistance of pH by buffers remains the same as desc ribed in the section. solution therefore contains undissociated acetic acid molecules. have increased the pH.---> CH3COO.. dissociates completely into its constituent long Na + a nd OH-. and Na+ long. The extra N H long ue to the dissociation of NH CI depress the dissociation of NH.---> CH3COOH + CIIn solution HCI dissociates completely to produce hydrogen long and chloride lon g. Let us a system of acetic acid and sodium acetate. CHsCOOH. bUt in the buffer solution they react with CH3COOH to giv e to water and acetate long. solutions containing weak bases and their salts are also buffer solu tions) because []/have the capacity to resist changes in pH when confronted with either an acid or a base. NaOH. we will consider it once again in a more explicit manner.+ Na÷ + H20 The strong alkali. The pH does not increase appreciably (it increases on ly in . . When an alkali (NaOH) is added : CH3COOH + Na÷ + OH. to the change in the ratio of acid to salt in the solution). . The salt diss ociate completely. The pH changes merely to 4. Similarly. the p] increases by 4 points and becomes 11.water (pH 7. an d its salt by the genen formula BA (B÷ being the metal ion and A. Solving for {H÷]. we get . The equation is d erived in following way. Similarly. while the weak acid dissociates only partly. Let us denote a weak acid by the general formula HA.1 N HCI is added to 990 ml of pure.01 units on the pH scale. Buffers are mixtures of we ak acids and the conjugate bases. We can write the equtli brium reactior for the dissociation of HA and BA in the buffer solution as follows : HA -I-I÷ + ABA -B+ + AWe will soon find that Henderson-Hasselbalch equation is simply another way or w ritir the expression for the dissociation constant of a weak acid. the pH of water drops 4 unil and becomes 3.75.76).01 points. the drop in pH is on] 0. The Henderson-Hssselbslch Equation Henderson-Hasselbalch equation is important for understanding buffer action and aci¢ base balance in the blood and tissues of the mammalian system. We thus see that buffer solutions resist chariges in pH to a very signific ant extent (we wi consider the same example quantitatively a little later).pH = pKa . if 10 ml of 0. However.77.1 NNaOH to 99 ml of above buffer solution elicits a rise of merely 0.0).log 24 Biophysical Chemlstr To what extent can a buffer solution resist change in pH ? A simple example will be citec If 10 ml of 0.being the conjugate base). addition of 10 ml of 0. The pH become 4. We have seen that the conjugate base provided by salt dissociation is actually i nvolved i the buffering action.1 N acetic acid and 0.1 N HCI is added to 990 ml of buffer consisting 0. We shoul therefore rewrite the definition of buffer solutions.1 M sodium acetate (pH 4. The metal long (like Na÷ in sodium acetate) are not involved . if 10 ml f 0.1 N NaOH is added to 990 ml of pure wate r. .Taking the negative logarithm of both sides.log Ka = pKa. the equation becomes However. . Therefore. and .log [H+] = pH. " has dissociated from BA and therefore the concentration of the conjugate base .?w .. is only slightly dissociated the absence of the salt. . Isalt] pH = pKa + Iog [acid] [proton acceptor] "'. the weak acid. the more accurately when concentrations are converted to . The value of pKa on the basis of activities can be calculaled with the help of relationship : and Bases 25 the negative sign. As with all the equations considered so far. Thus very little of the A.long. We can also assume that all . On the other hand. we invert . Taking into consideration these assumptioBs .or also [conjugate base] or pH = pKa + log [acid] necessary because the values of pK and activities vary strength. [A-] is equal the concentration of the salt. HA. [BA].long come through the dissoc iation of bak acid. therefore. be safely assumed that the co ncentration of he undissociated acid [HA] is equal to the total acid concentration.0n donor] .log [HA]/[A-] and obtain pH = pKa + log This is Henderson-Hasselbalch equation'Now. pH = pKa + log [prot. It can. he equation can take many differont forms. we have seen that the salt BA is completely dissoci ated and gives Ehlgh concentration of A. - . CH3COO. We have seen addition of 10 ml 0.1 N acetic acid and 0. . For most calculations. the drop in acetate ion concentration will be 10-3 mole/litre.1 NHCI to 990 ml of pure water brings its pH down from 7 to 3. Let us add this acid to 990 ml of 0.018 f Is the ionic strength of the solution.us see the quantitative basis of buffer solutions rslsting a large change in pH .1. that we have derived an equation which relates pH to concentration (conjugate base Concentration) and the weak acid concentration. concentra tions fairly accurate results.+H+ CH3COOH The addition of HCI therefore lowers the concentration of the acetate ion slight ly and the concentration of acetic acid by the same amount. Now.1 M sodium acetate long disgociating from HC1 are neutralized by the acetate long. If we assume that all I-I+ long have neutralized. The concentration acid would rise by the same amount.pK (activity) = pK (concentration) . however. lactate buffer containing 0. It will be equ to the pK of the acid. the actual salt and acid concentratior can be varied widely without any change in pH if the ratio between the two is un ity.i mole mole mole -.0. In actual cases. The lowe r the pK oft] acid the lower will be the pH.101 pH = pKa + Iog-pKa of acetic acid is 4. Biophysical Chemistr [CH3COO.001 = 0. according to Henderson-Hasselbalch relationship.1---0.01 M lactate and 0.76. Therefore.0999 0. This increase is not signii2cant enough.0 pH = pK + 0 pH = pKa Thus to calculate the pK of any acid one only needs to dissolve that acid and it s salt equal concentrations and thenaexperimentally determine the pH of the solu tion.0999-NAL litre litre litre . the pH of the dilut buffer increases slightly.01 N lactic acid will have the sa me pH even if fl buffer is diluted 10 times or even 20 times. however. Some extremely important problems ab out buffers which can be solw using Hederson-Hasselbalch equation are provided f or in Box 1.6. Henderson-Hasselbalch equation makes it clear that the pH of a buffer solution d epen{ upon the pKa of the acid and upon the salt to acid concentration ratio. Thus. pH = pKa + log 1. The buffer pH will increase with increasing salt concentratio Again.26 0. Henderson-Hasselbalch equation gives a very important relationship which makes possible to calculate the pK 0f-any given acid with extreme ease.= 01101 litre litre litre Substituting the final salt and acid concentrations in the Henderson-Hasselbalch equatio we get pH = 4.mole mole mole j[CH3COOH]FINAL = 0.+ 0.0999 0. [salt] !i.001 -.76 + log 0.75 The pH of the buffer solution after addition of I0 ml of 0.: ". that if tt molecular ratio oLIt to acid is unity in a solution.76 to 4. the pH of that solution wil l be equal to tt pK of flleacl tTind "< : ::.751 drop of merely 0.. pH = pKa + log [acid] . The relationsh ip is.r the ¢.1 -.01 units of pH.1 N HCI changes from 4.I01 = 4. 86 + (0.) the change in pH of the buffer solution is therefore 3.09 pH unit.0414) = 3.86 + log = 3.86 .3.09.025 M I and I0 ml of 0. T he ca to resist changes in pH depends upon (i) the actual concentrations of salt and a cid pre¢ the buffer.49 the change in pH of the buffer solution is therefore 3.49 = 0. i n this c ml of 0.1. sin ratio of salt to acid is unity). What will be the change in pH ? The HCI.025 N lactic acid.1 N lactic acid {pH of this buffer will be equal to the pK. The pH of the solution will therefore be pH = 3. .1 N HCI to a buffer solution containing I0 ml of 0.3. in th is cas convert 4 ml of salt to acid.1 N HCI to a lactate buffer solution containing 10 ml of 0.of lacti c acid.1461) = 3. Suppose we add 1 ml of 0. This means that buffers containing higher concentrations of salt and aci d have a buffer capacity as compared to solutions with lower salt and acid conce ntratns.77 (App.86 + log 6-1og 14 14 = 3.37 pH unit. o ne ml of HCI causes a decrease of about 0.7782 . 1 M lactal 10 rnl of 0.9542 1. The pH of the solution will therefore b 9 pH = 3.28 Btophys/cal Chel Buffer Capacity By buffer capacity we mean the capacity of the buffer to resist changes in pH.86 + log -6 = 3.86. Thus. 3. What will be the change in pH of thebuffer solu tion ? The H convert 1 ml of the salt to 1 ml of acid. ' First.86 . The above example tells us that the first buffer has a higher buffer capacity th a second.37. let us consider the effect of actual salt and acid concentration on the buffer ca Let us add 1 ml of 0.76 = 0. Thus. and (//) the salt to acid concentration ratio.86 + (0.1 N HCI causes a decrease of about 0.86 + log 9-1og 11 II = 3. The pH of this buffer would be pH = 3.1 . The HCI would convert 1 ml of salt to 1 acid.86+ (1:1461-0.23 6 Thus the pH of the buffer is lowered by 0.86+ log 14 = 3. The generalized statement based on the above example can be that when the ra salt to acid concentration is unity.34 5 Let us now add 1 rnl of 0. This example elaborates the effect of salt to acid concentratio n ratio on b capacity.1 h added to a buffer composed of 10 ml of 0. The pH of the buffer will be pH= 3.Let us now consider the effect of salt to acid concentration ratio upon the buff er cap To understand this.176-0.86 + log "" = 3.1 N HCI to this buffer.1 Nlactic acid changes it by 0.6989) = 4. . As shown previously. let us consider a lactate buffer composed of 15 ml of 0. the buffer has maximum efficiency.1 N lactic acid.09 pH unit.1 M lactate and 10 ml 0. 1 ra l of 0.7782)= 4.1 Ml actate ano of 0. pH unit.86 + (1. lactate buffer should be a good buffer in the pH . For the pH range 3 to 4.5. Buffer The advantages of phosphate buffers are numerous.these inhibition of some enzymes. 2.86.chemical characteristics p eculiar which must be borne in mind. It consists of one pH unit on side of the pK of the buffer acid. However. PRECAUTIONARY INFORMATION ABOUT COMMONLY USED BUFFERS As mentioned earlier. How important buffers are for normal functioning of a body can be . In the laboratory. A few most commonly used are discussed below individually. Iwe increase the concentration of buffer solution. high ionic strength can be obtained without the . each buffer has other. Thus. precipitation of polyvalent cations. They have a high buffe ring capacity. The selection of a proper buffer system for a given experime ntal a common problem.that the pH of blood is maintained strictly within the range 7.86 -. absor ption light. The most common problems that plague . and (//) to maintain optimum acid reaction of a medium such as bacteria or tissue culture or an enzymatic rea ction mixture.9.4.understood fr om the .3 to 7. discuss more about some important biological buffers in a later s ection. Na and K salts are very highly soluble and thus any ratio of Na÷ and K÷ long can be Because the long are highly charged. toxicity. Several buffers may fit the pH range one is working in. strong effect of concentration and temperature on pH.and Bases 29 The buffer range of any given buffer is about 2 pH units. a few of them may have characteristics that are detrimental to the experimental This becomes even more important considering the fact that most of the commonly used designed for biochemical use. buffers are used for two mai n : (/) as reference standards for pH determination. Death is more or less pH of 7. phthalic acid phthalate can be used. it is the pK value that is of utmost importance when decid ing about buffer has to be used. and lack of good activity in the most used pH range in biochemistry. for the pH range 4-6.0 and above a pH of 7. Some examples arc provided. acetic acid-sodium acetate is satisfactory. monosodium dihydrogen phosphate (acid ) monohydrogen phosphate (salt) buffer is useful (see Appendix). for the pH range 6 t-0 8. we can also i ncrease its to a little extent. More importantly. The last named advantage can become a disadvantage too. advantages. The buffering range in which these buffers work well is 10 . They ma y bind polyvalent 2+ + Chiefly. they bind Ca .8.5.molarity.5. Mg2 . They are good buffers between the pH range 12. Another disadvantage isthe lac k of buffering in the range 7.0. .5 to 8.12. 5 and 8. The principal disadvantages of these buffers result because of relative insolubility of most of the sensitivity of pH to temperature chauges. C onsider . it has a high buffering capacity between 7. It is impossible to prep are a buffer with a high buffering capacity and a low ionic strength! The actual disadvantage of the phosphate buffers are as follows. and to a lesser extent.0 .10. phosphate are known to be toxic to mammalian cells. (1) Since the pKa is . Buffer This buffer is probably the most used in biochemistry and for obvious reasons. High temperatures extreme pH changes due to loss of CO2. Borate is good between pH range 8.001 M). . Both these buffers ha ve a I UV absorption.7 and glycine between 9. As such. Another buffer that suffers from high absorbance in the UV range is the barbitur ate Boric Acid and Glycine Buffers Borate has weak toxicity and glycine. the reason is that Mg2÷ is a cofactor for nucleas es of EDTA therefore abolishes the activity of these enzymes. (2) Like phosphate buffers. Additionally.0. This is another major plus since enz yme assays in the UV range will not be impeded. One precaution here. ( 4) pure forms. These are precisely the cations that are used very for enzymological work.30 Biophysical (2)Very low toxicity. the concentration of this buffer should be kept yery low (0 . One more great advantage is that it has for the divalent cations Ca2+ and Mg2+. of course. This is exactly why E DTA buffer used when nucleic acids are to be stored. (3) Does not intei-fere with most biochemical reactions. Thus. one finds that EDTA buffers are us ed very when working with nucleic acids. The disadvantages are as follows: (1) Like carbonate buffers. 5 to 10. EDTA suffers from disadvantage of absorbing very highly in the UV range. The disadvantage with glycylglycine springs from its being a peptide : it is . its to a very high extent. Ag+ etc. It also has very low UV absorbance.7 to 9. It is a chelating agent of divalent cations and is added to other buffers mainly to redu ce concentrations of the divalent cations. borate is chosen for work with bacteriophages since it stabilizes Glycylgly¢inc Buffer This is often a buffer of choice for enzymo10gical work and works well in the pH range to 8. (3) It reacts with some glass electrodes and thus may lead to err oneous EDTA Buffers EDTA (ethylenediaminetetraacetate) is not normally used for its buffering. if nucleic acid has to be estimated. it reacts with a few metal lo ng like Ni2+. has none. their full names are being provided in the table. These buffers are given bel in a tabulated form (Table 1. They are not toxic. Good looked at a large number of zwitt erionic buffer The buffers that he found good lack the drawbacks mentioned above. it cannot be crude protein preparations since such preparations may have protease contaminati on. Triethanolamine Buffer This is another favorite for enzymological work.proteases and as such cannot be used with these enzymes. .7). namely protease susceptibility. they are usual ly known their abbreviations. The Good Buffers These buffers are so named after their discoverer. their pH not sensitive to temperature changes. they do not precipitate divalent cati ons. Additionally. it is a volatile buffer and therefore may chosen for purification work where the buffer is to be subsequently removed. the. It buffers at the same pH range and it doesn't suffer from the li mitation glycylglycine. However. It has all the advantages glycylglycine. and they are quite soluble. Norman Good. Also. Since they have very long names. Because of sever problems with the buffers just discussed. do not absorb appreciably in the UV range. 2 and Bases 1.88 6.31 ethane acid ADA 6.lycine propaneacid -hydroxyethyl). Good's Buffers Buffer Abbreviation pKa pH range where (20°C) best used -2.7.I -piperazineacid ethanesulfonlc .2 .7.7.4 . acid iminodlacetic [(carbamoylmethyl) imino]acid acid .62 6.4 acid.ACES 6. 80 8.15 7.O 7.35 10.8 9.6 .1 9.1 8.2 8.20 6.5 6.O 8.8 - 10.6 6.8 .5 7.9 7.O 7.55 81o 6.6 7.6 5.8 6.8.BES Bicine CAPS CHES HEPES HEPPS* MES MOPS PIPES TAPS 7.8 .7.15 8.55 7.4 7.40 9.11.7.40 6. Neutralization need not result in the formation of a recognizable salt a nd may involve water. . TITRATIONS THE INTERACTION OF AN ACID WITH A BASE The old definition of neutralization states that an acid and a base react with e ach other to water.8 known as EPPS.acid 1-bisglycine TES Tricine 7.0 .8.6 . neutralization is a process of proton transfer from an a base.0 7.15 7.8. The Bronsted-Lowry concept offers a much broader view 3333of the process of to this concept.50 8. As soon as the fr H+ is neutralized by OH. before any NaOH is added). Figure 1.7 represents the characte rist titration curve of acetic acid when it is titrated against a strong alkali. in the following pages.to water.32 HA ÷ B (acid) (base) Biophysical Chemistn.e. we shall be considering acid-base interactions in aqueot media. When successive aliquots of NaOH are added . BH+ + A- (conjugate (conjugate acid) base) Although. the above discussion will help us in identifying the conjugate acid and b ase produced: any neutralization process. Small aliquots of the base are added till the acid is total ly neutralized. The figure traces fl course of titration of a 0. some of the undissociated acetic acid immedia . the acetic acid is slightl y ionized and the I: of the solution is due to acid alone. Titration Curves of Weak Acids Let us again take the example of acetic acid. Titration is normally used to determine the amount of an acid in a given solutio n. titration can be followed by adding an indicator to the acid solution or by cont inuo measurement of the pH by a pH meter. the OH= fro] dissociation of NaOH will combine with the free H+ in solution to form water. T. In th procedure a known volume of an acid is titrated with a base (usually NaOH) whose concentrati( is accurately known.1 N solution of acetic acid with 0o I N NaOH at 25C. The concentration of the base and the volum e requir¢ for fully neutralizing the acid are sufficient for calculations which will revea l the concentratic of the acid in solution. Before tl titration is started (i. If we plot the pH values against th volume of alkali added we get the characteristic curve shown in Figure 1 . i. Th the pH of the solution when the acid is half titrated represents the pK of the acid being titrate (Figure 1.e. Thus with each addition of NaOH.7). Thus .b y pH 7.----) CH3COO. Such a situation will clearly be present at the mid-p oint of the titration.ation curves of these two acids will be displaced along the pH scale accor .. We know th solutions of weak acids and their conjugate bases are known as buffers. at any stage of titration. and the we acid. If the pK of the acid being titrated is the titration begin's at pH 5. acetate. With the progress titration. They differ only in t heir location on the pl scale. for weak acid whose pK is 5. Th tiI4". CH3COOH + Na+ + OH. is half completed. we havre alre ady considered th the pK.7). m ore water formed and more and more acetic acid gets converted to the acetate anion. This acid will be half titrated pH 5 and will stand c%mpletely titrated at around pH 7. aad is complete at around pH 9. acetic acid. we should be able to calculate the pH ofth solution using the Henderson-Hasselbalch relationship. Have we come across this situation before ? Yes. The position of the curve on the pH scale depends upon the pK of the acid being titrate( While dealing with the Henderson-Hasselbalch relationship. The pH of this solution will now change in accordance with the Hendersor Hasselbalch equation.+ H20 + Na÷ As the titration progresses.7. the titration begins at around pH 3. the solution is fast becoming a mixture of the conjugate base . The titration curve of all weak acids have similar shape (Figure 1.tely dissociat( further to satisfy its dissociation constant. a The titration curve of a weak acid is usually spread over about 4 pH units. the concentration of acetate ion increases continuously am that of acetic acid decreases. of an acid is equal to the pH of the solution containing equal c oncentrations of both salt anl the acid. ding to their respectiv pkg. . 0 . zones: Acetate 3 2 1 O 0.and Bases pH 7 33 !4 13 12 11 10 9 I Midpoint titration Efficient : 9-2S buffering .5 1. Titration curves of weak bases follow the same pattern as seen for weak acids.7 also shows that at both the. This means that the composition of the acid-conjugate base solution in these is not good foi. On the of these curves one can select the salt acid concentrations that will give a goo d buffer One can see that the titration curve assumes greatest degree of flatness at its pK the acid to conjugate base concentration ratio is unity. This is the proof for what we have already considered mathematically : the buffer is most efficient in resisting pH changes when the ratio of salt is unity.7. . midpoint and end o f the titrations. Also indicated are the predominant ionic species at the beginning. Figure 1. ends the titration curve sharply. This ratio obviously ha s the capacity.7). b ut in a order as evident from Figure I. From Figure 1.8. The midpoints of the titr a(-ions have been indicated.7. zones are the buffering regions of the acid-conjugate base pair (Figure 1.a buffer.Equlvalente of OH------1. It is obvious that at both the ends the ratio of conj ugate base to is far removed from unity. The buffering zones have been shown. we note that the titration curves are relatively flat in their centre sections. Characteristic titration curves of weak acids. 9.8.1 N .0 Equivalents of OH100% 0.1N NaOH Salt lgure 1.34 2 I of titration 11 I 10 NH4CI pkw" pkb = 9.5 1.HCI Figure 1.1 N HCI against 0. Characteristic titration curve of weak bases.26 = PH 7 r I 1 10 0 0.0 -----. Titration of a strong acid with strong base (0.5 1. Titration of a Strong Acid with a Strong Base 4 pH 3 OI Acid 0. for which three ionization steps and there corresponding pKa are . almost 80% of the NaOH is required.9 represents the titration curve of 0. To change t he pH by one unit. titration curves of strong acids are not so important to a biochemist. Titration Curves of Polybasic Acids Let us now consider titration curves of polybasic acids which can donate more th an one proton and can consequently possess more than one pK. A good example is affored by phosphoric aci d. H3PO4.1 N Na OH. As this pH range is seldom used in biology. at the later stages the titra tion curve shows a sharp break and the pH changes rapidly.1 N HCI titrated against 0.NaOH). The Striking thing about this titration curve {in general for titration curves of al l strong acids) is the very sluggish change in pH as successive aliquots of NaOH are added. corresponding to the succe ssive dissociation of each of the protons. Figure 1. Thus the HCI solution has a good buffe ring capacity between pHI and 3. However. 1 N H3PO4 solution by 0. Citric a cid has values which are relatively close to each other (pK1 = 3.and Bases 35 I-13PO4 H2PO +H+. groups enter into multistep acid-base processes closely analogous to those of ph osphoric 11:[ orHPO--.7. the titration of the second H* is not complete before the t hird H* In such cases there are no sharp breaks in the titration curve between successiv e and one observes a relatively flat curve throughout. there are three different p H zones at which hosphoric acid system can act as a very good buffer. .24 H2PO HPO+ H+. Three buffering zones. However. This example of phosphoric acid has r chosen because many biological molecules contain phosphate-related groups.2 HPO. pKa = 2.PO +H÷p/G=12.7. What happens if the polybasic acid happens to have different pKa values very clo se to each Let us answer this question with the help of an example of citric acid.PO--'+ H+.75 ml of 0. Likewise. are self evldent. case what will happen is that by the time the first H* is fugy titrated the se cond H* also titrating. and the third in titration of HPO : to phosphoric acid the three pK are much separated from each other.----for H.P4 HPO. If acids having pK= values not far away from each other are mixed together.50 . pKa of three different stages are shown. 8t .1.4 Thus. pKa = 12. therefore.1N NaOH Titration of 25 mls of O. pK3 = 6. In . one wou .2 * p 2 24 0 25 -. shows a sharp break after each pK and at these regions the buffering capacity 'the solutions is very poor (Figure 1.1 N NaOH solution.4 . I0). in a titration of phosphoric acid the first stage consists of titration of H3PO¢ to the second in the titration of H2PO to HPO -. This is evident from the titration curve of citric acid shown in Fi gure 1.+ H+p = 7. pKa . although not shown. The titrat ion.4 ).11. Such systems are well buffe red over a range of pH. pK2 = 4. Their titration curves therefore thereby enhancing their efficiency in the pH range maintained by the body fluids . This is what happens in the body. The pKa relaUvely close to each other.ld similar type of curve for such a mixture also. . I I. Citric acid therefore has a large buffering zone. We will also discuss in brief the pH-dependent properties o f o biomolecules. It is the refore a obvious conclusion that biomolecules are profoundly affected by changes in pH. I n any c most of the important conponents of the living cell are acidic. and thir d stages of hydrog dissociation. . Ionization of Amino Acids is pH-Depenflent All amino acids are amphiprotic compounds and can be denoted by the general forn R I H -. tissue cultures and bacterial cultures in inadequately buffered media. I O. Compare thi s cun with Fig. or amphot eric and alteration in the pH of the environment profoundly affects their state of ioniza tion and the: their conformation and biological activity. Citrate ti gives a characterlstlc jlat curve because of overlapping flrst. Examples may also be cite d of dea. amino acids. FUNCTION AND STRUCTURE OF BIOMOLECULES IS pH DEPENDENT The death of a human being below a blood pH of 7. In this section we will deal with pH-dependent properties of proteins and their buff blocks.C k NH2 COOH Their a-amino group is weakly basic and has a pK in the range 9-I0.9 is en testimony for the importance of pH to life in general.5. basic.36 Biophysical Chemt 13 12 II i0 0 NaOH Figure I. I. second. Titration of citric acid by NaOH equivalent strength.0 and above a pH of 7. The amino acids which do not possess any dissociable group in the side chain exi three ionic forms : R R R H-.H -.7 to 2.4.a a-carboxyl is acidic with the pK in the range 1.C--NH2 COOH CO0COOCATION ZWITrERION ANION .r a..NH r . H--C -.C --NH . All amino acids are th erefore ioniz a an aqueous solution depending on the prevailing pH. pH= 2 E/iv of O H--curve for alanine. Similarly. The charges cancel o ut the amino acid possesses no net charge.C -. at pH equal to the pK of the ami no the amino acid will exist partly as anion and partly as zwitterion. At a low pH only the a-amino group is ionized and the amino acid is a cation. partly zwitterion. If the pH is still further raised.NH2 COOH zwitterion form in solution will be CH3 H-C --NH COO- . amino acid may be called a zw/tter/on. Let us take the example of a lanine. If the pH is a-carboxyl group starts dissociating. Thus. the hydrogen ion from group dissociates. pKal is for -COOH andpl is for the a-NH . This leaves only the negative charge on the amino acid due to the dissociation and the amino .acid behaves as an anion. CH3 H-. In a solutio n in water the amino acid exists mostly as a zwitterion. This process leaves a hgative charge on th e which already has a positive charge due to the amino group. This state is known as the zwitterionic state and . on the basis of the principles we have discussed earlier (Henderson-Hassel balch it can be said that at a pH equal to the pKa of the carboxyl group (PKal) the am ino as partly cation. From these two pKavalu es we can calculate the pH of the solution of alani ne in its zwitterionic form by the equation . The re action can be represented by the equation " CH3 CH3 +H3N.H2N--CH--COO. if an alkali is added. Prom the midp oint of the first titration curve we can calculate the PKal (for the | dissociation of carboxyl group) and from the mid-point of 1 the second titration curve we can calculate the pKa2 [fo r the * dissociation of the amino group).CH. HCI. to this solution of alanine in water it will behave as a base. IK. a-carboxyl group as a base.COOH + Clthe other hand.+ Na÷ + H20 in the zwitterionic alanine. a-amino group behaves as an acid and the .+Na÷+ OH.12 shows that the titration curve for a lanine looks like that of a diprotic weak acid.+H3N.COO.CH.+ H÷ + Cl.acid. What would the titration curve of alanine look like? Figure 1. The reaction can be expressed by the equation ÷HaN--CHCOO. alanine solution behaves as an acid. when we dissolve crystalline alanine in pure water.x CHa CHa =9.02 2 Thus.69 pH = = 6.02.CATION ACID 38 Thus for alanine Biophysical Chern 2.34 ak-------. In general the pH at which an amino acid exists as a zwitterio] known as the isoionic or isoelectric point of that amino acid.. alanlne wI exist as a zwitt erion. This p} known as the/so/on/c po/nt of alanine as alanlne at this pH does not possess any net chin This pH is also known as the tsoelectrtc pH of alanine as at this pH alanine swi ll electrophoretically immobile. We may now write the ionization of alanine indicating the specific pK ' CH3 H -.34 + 9.NH .69 PKa H C -. Or if an alanine solution is brought to the pH 6. the pH of the solution is 6.C NH3+ COOH PK =2. Their isoior points and different pKa are also shown in the figures. Equivaleflla of OH: EClUiValenl of OH- Figure 1. as evident from the fig ures. for -COOH. The.. Table 1.13 and 1.. glutamic acid) while some others might contain extra amino group (e.Their titration curves look like those of polyprotic acids. pK. lysine}. Titrati( curves for aspartic acid and lysine are given in Figure 1. groups might be acidic or basic in character.g.g. The student can thus wri te th< dissociation sequence from the data provided in the figures ( the sequence would be much li that of alanine represented above). Tltratlon cw for aspartlc ac pK is for a-COOH. some where arour 10. pl¢ for a-NH 3 ' and pK .. aspartic acid. pK= is fo= -COOH. It may be mentioned... Titration curve forlysi+ne.14 respective ly.8 lists the pKa and the pI values of important amino acids. while the isoion pH for the diamino-monocarboxylic amino acids such as lysine is quite high. that tt Isoionic pH of dicarboxylic amino acids are quite low somewhere around 3.C --' NH3÷ COOCOOzwrITERION ANION pH 6.14.02 ALKALI ISOIONIC POINT There are many amino acids which have side chains containing dissociable groups. Some of them might contain an extr a carboxy] group in their side chain (e. and pl for Figure 1.13. 71 8.82 6.78 2.36 9.17 2.04 12.48 2.69 2.09 3.36 9.0 9.18 8.6 1.33 10.25 9.86 9.95 .67 2.34 9.60 2.68 2.17 9.39 Cysteine Isoleucine Leucine Lyse Proline PKa 2.19 4.35 9.82 1. 21 1.53 2.10.75 5.98 6.58 6.68 and Bases .98 5.97 7.22 5.15 pI 6.02 10.99 10.83 9.60 2.76 2.98 5.13 1.02 5.21 9.28 9.02 3.10 5. a-COOH -COOH a-NH a-COOH a-NH -SH a-COOH ¥-COOH a-NH a-COOH a-NH a-COOH Imidazole-NH+ a-NH -COOH a-NH a-COOH a-COOH a-NH -NH a-COOH a-NH a-COOH a-NH a-COOH a-NH a-COOH a-NH .-COOH a-NH a-COOH . a-NH Guanldtnlum-NH.1.8 pKa and pl Values of Some Common Am/no Acids Conjugate acid Aspartic acid Glutamic acid Glycine Hisdine Methionine Phenylalanine a. 32 9.65 5. Its imidazole is half ionized at that pH. We have alre ady considered importance of pKa for the buffering action of solutions.88 5.8 it becomes clear that the only amino acid which has a p/Q near to the pH of blood body fluids is histidine.62 What is it that we learn from the acid-base properties of these amino acids? 1.07 2. Because it has a pKa ne ar to the .63 10.11 10.43 2.97 Trytophan Tyrostne a-COOH a-NH ct-COOH a-NH a-COOH a-NH --OH a-COOH a-NH 2.4O Threonine Valine 6.39 2.53 5.20 9.38 9. protein of erythrocytes is a unique protein in that it contains a large number o fhistidine in its structure. which is important to the role of red blood cells in the transport of oxygen carbon dioxide by the blood. . histidine possesses a significant buffering action in blood. These histldine residues impart considerable buffering power t o haemo near pH 7.PH. We can cite the example of solubility of amino acids. . the ultraviolet absorption at a given wavelength and even the biological Can be Separated on the Basis of Charge Can we utilize this pH-dependent ionization of amino acids for their separation? A small are given a mixture of three amino acids. Thus. Amino acids least soluble at their isoionic pHs. metal c helating optical rotation. alanine. at a given pH different amino acids will have different solubilities upon how far removed is the pH of the solution from their own isoionic points. aspartic It is required thatwe separate these three amino acids from each other and /hem in a more or less pure state. Other properties which are affected by the extent of ionization are the.Bases 41 of Amino Acids are pH-Dependent "All the properties of amino acids which depend upon their ionization would natu rally be prevailing pH. From Table 1.8 we can find out that the pl of alanine. They are much more soluble at pHs lower and higher pHs. 15). There are. We. however.r) that ionization of proteins should also be pH-dependent since proteins are amino acids Itnke together by peptide bonds.1 COO" ginin Cim Figure 1. Lysine on the other hand wi move towards the cathode and alanlne will remain immobile because it contains no net char (Figure 1. It is therefore a c oro.COOI Aepertio Imid CHNI+ Net hargo zero Itet charge. the basis of the differential charge they possess at different pHs. argnlne becomes positively charged and moves toword the cathode. vz. free a-amino group a the amino end of the protein.successive amino acids are involved in the formation of pep tide bon (Figure I. defer a discussion on this tech nique to later Chapter (chapter 1 I) where it is discussed in reasonable details . If the pH of a solutWn of th ree arnlno acids.a. separated by paper electrophoresis using high voltages (see chapter 12). aspar acld and argIne s brought to 6. aspart acid will move towards the anode by virtue of its negative charge.. We can thus effectively separate a mixture of amino acids by elec trophoresis o. Amino acids are usual. Ionization of Proteins i pH-Dependent We have seen that ionization of amino acids is pH-dependent. Thus. 16) they ar unavailable for ionization. If a potential difference is now applied across the vessel containing this solut ion.15. only the terminal. But since a protein consists of manyamino acids possessing dis . Since th e a-amlno an¢ a-carboxyl groups of. however. A better method to separate amino acids . pH dependent separutWn of amlno acids.0. due to ther different pK values alantne becomes zwltterlon the anode. significant differences. and the free a-carboxyl group at the c-termlnal en d of the protek remain ionizable.on the basis of the charge they possess at differen pHs is ion exchange chromatography. Now if we bri ng the pH off solution containing a mixture of these three amino acids to 6. 42 Biophys. of these amino acids? Since the pH is extremely near the pI of alanine.76 respectively.0. 2. the ionization of proteins also is pH-dependent.98. and arglnine are6.sociable side chain groups. and 10.02. Aspartic acid on the other hand will be negativ( charged at this pH and arginine will bear a positive charge. these dissociable groups will provide a protein with its character istic charge pattern at a given pH.d Chemist aspartic acid. this ami no acid will present as a zwitterion with no net charge. what will be th e state ofionizatl. I I . As we have seen that the dissociation of these side chain groups is also pH-dependent. C-NH imidazole pH 12 .I.NH2 -COO- .NH2 .(CH2).NH CO0CH2.(CH2).C.and Bases 43 (ore-) pH7 -1 protein NH= .NH.N -Nll tl (CH2)-NH.COOH÷ . Since the numb er of the ioni groups and the extent of ionization both are a function of changes in pH.' characteristic feature of the curve is that it does not break sharply at any pH and is relati. Dissociation curve of standard gelatin preparatR)n in hydrochloric . Proteins Can Act as Buffers Figure 1.44 Biophysical Chemts The Three Dimensional Protein Structure is pH-Dependent The conformation of a protein molecule in space is a result of a specillc coilin g and fok of the polypeptide chains. due to the multi dissociab!e character of the protein. The molecule may be very large with molecular weight extendin several million daltons. We will discuss ra about proteins as buffers a little later. Example may be cited of myoglobin in which all the polar side chain am ino acids clustered at the periphery of the molecule and the non-polar amino acids are clu stered centr The core of the molecule is therefore hydrophobic while the periphery is hydroph ilic. 17. wl other amino acids with non-polar side chains are clustered together in another p art of molecule.17 shows the titration curve of protein taking gelatin as an example. The absence of sharp breaks and the relative flatness at many p H range. Most globular proteins fold themselves up in a manner that most of the amino aci ds v potentially ionizable polar side chains are clustered together in one part of th e molecule. Other types of bonds such as the electrostatic bonds between ionized gro of opposite charges are important for maintaining this stability.10) . flat at many pH ranges. This type of a titration curve is a hallmark of a good b uffer with a I buffering range. The stability of the three dimensional structure is not entirely du the peptide bonds. There are many ionlzable groups wh ionize successively and simultaneously throughout most of the pH range. 123456 78 910111213 pH Figure I. the st ability of three dimensional structure of proteins is also pH-dependent (also see Box 1. acid or sodium hydroxide solutio 30°C. Since this is an ideal condition and not usually obtainab le. The isoelectric pH of a protein is the pH at which the protein is immobile in an elecl field.. the isoio and isoelectric points of a given protein differ. i. Isoionic and isoelectric point of a protein will essentially be the same if the protein does bind long other than H÷.e. on the basis of the charge density they possess. the net charge being zero. The isoionic point of a protein is defined a s the pH at wh the total number of H÷ taken up by a protein is equal to the total number of H÷ diss ociated fr it. . At this pH. i n an electrophore .Separation Methods for Proteins Depend on Ionization Just as discussed for amino acids. the protein exists as a zwitterion having equal number of pos itive and negal charges. Thus. proteins are also separated on the basis of t he extenl their ionization. Even slight changes in pH . A much better technique of protein purification is that of isoelectxic focussing . the desired protein will become immobile since it possesses charge. crcnt proteins :with differing mobflities because their charge density is different and become separated each other.10). This was primarily the evidence which led to the suggestion that the activity an attribute of a small part . any change in pH. . We defer a discussion of this technique to Chapter 11. the pH is isoelectric for it. of Proteins is pH-Depenflent We have already seen that the three dimensional structure of proteins is depende nt upon stability. while r proteins will continue moving. Use is e of the isoelectric point of a protein. Thus. This is the reason why slight alterations in pH cause a change in the acti vity of a may also be mentioned that the effect of pH on the biological activity of protei ns may evidence about the dissociable groups present in an active site. Another powerful tool for protein purification which also depends upon the charg e density ion-exchange chromatography.of the whole molecule. which is to the three dimensional structure of protein molecules is definitely detrimenta l to We know that enzyme activities are a function of pH of the medium. At the space where pH of the gradient is 6. The method is discussed in reasonable deta ils in 12. all the proteins will mig rate the charge that they possess. and in general. the active site (the site is engaged in catalysis). Suppose that the isoelectric point of a protein is 6. alter the activity of an enzyme in particula r.0.and Bases 45 the mobility of a protein is a funcUon of its charge density. Biological activity of proteins is known to be an attribute of one sp ecific of the protein molecules (also see Box 1. At extreme pHs the might become denatured losing its function completely. a pH gradient in a column so that the pH varies from 1-10 and place all the the sample on top of the column and start the current. r not be of a destabilizing nature. Small pH changes which might not alter the structure o f a substantial enough to alter the ionization of a single dissociable group within this site. Large from optimal pH alter the activity of an enzyme drastically. The process is discussed in details in Chapter 12. . 46 Biophy . 0. be expected.1 us. general acid-base catalysis is. the strength of the general acid or base. How can we ascertain that a proton transfer is the mechanism behi nd can carry out the reaction (which we suspect to be acid-base catalyzed) (heavy water) rather than H20. To hydrolyze bonds without enzymatic mediation. Due to isotope effect (see Chapter 13) deuterium ion will reac t much ' than the hydrogen ion. The imidazole is particularly effective in this case also. very high concentrations ofH+ and OH. "ff the reaction is catalyzed by a proton transfe r step. can be proved by a simple example. The most significant at physiological pH is the imidazole group of histidine which has a pKavalue 6. Chymotrypsin achieves this hydrolysis pH and at body temperature fairly quickly. its proton dissocia tion of reactions catalyzed by proton transfer steps. It has an equal rate of protonation and physiological pH with a half-time of less than 0.are req uired long reaction periods. i. This pKa enables it to act both as a proton donor and a proton acceptor at pH. !take place at a much slower rate in heavy water.Bases 47 Catalysis In reactions catalyzed by general acids or bases the catalyst functions as an ac ceptor or protons. There are several enzymes catalytic properties to the tmidazole group of histidine. Deuterium is an isotope of hydrogen and has twice as mass as hydrogen. of Most Biomolecules is pH-Dependent . Thus. the acid donates a proton to the reaction interm ediate he base accepts one. Many times both a general acid and a base are involved in catalysis.. Using such strategies many enz ymatic have been shown to be acid-base catalyzed.e. Another factor which can affect the rate of acid-base catalyzed reactions is or accepts a proton. Often the critical proton transfer step is to or from a carbon atom of the state species. sess multiple dissociable groups will be affected by changes in similar to the proteins described above. t only biopolymers but even smaller metabolites may be affected by pH. One of th e main t of the cellular metabolites being charged is that the plasma membrane is lmpex?meable to charged species. will be affected by changes in pH. Their three dimensional structures and their biological activity would be affected by changes in pH. Important ionizable groups possessed by cellular metabotites . Thus. nucleic acids.which pos. It is therefore easier for the cell to retain t hese the cell. mucopolysaccharide s. A detailed description of haemoglobin is uncalled for here. phosphate buffers and protein/proteinate buffers. In this process the oxyhaemoglobin becomes deoxygenated and via venous blood retu to the lungs to indulge in the oxygen transport cycle again. We therefore grasp the importance of maintenance of the pH of physiological fluids within limits conducive for normal functioning of cellular processes. (//) the acid and base s pecies oxygenated haemoglobin. The predominant function of haemoglobin is to tran.4 for venous bl¢ and 7. The oxygena haemoglobin. while it is being carried by the blood. While engaging thus in its prim . The control of pH in biolo systems is achieved by the action of efficient buffering systems whose chemical nature that they can resist pH changes due to the metabolic production of acids such as lactic a and bases such as ammonia.38-7. Within the lungs. The major buffering systems found in cellular fluids are I bicarbonate buffers. The pH here is maintained within extremely narrow limits of 7.36-7. and the phosphate group. Biologically Important Buffers We have seen how all major biomolecules are affected by pH changes.¢ oxygen from lungs to respiring tissues. donates its oxygen to vario us respi tissues. Buffering of Blood An interesting example of buffering in biological systems is provided by what oc curs blood. Even the rate of catalys is metabolites may be dependent upon their extent of ionization and will therefore be upon pH.48 Biophysical are carboxyl and amino groups. but what can be ment ioner that this iron containing protein resides within the red blood cells in relative ly abundant quant 14-16 grams/I00 ml of whole blood. oxygen from the incomi ng air ent the erythrocytes and becomes bound to the iron atoms of haemoglobin.42 for arterial blood. and (///) the acid and base species of deoxygenated haem oglobin. Three distinct buffering systems are responsib le for maintair the pH within these narrow limits : (/) H2CO3 and HCO . The conclusi on we have derived is that the biological activity of most biomolecules is dependen t on pH. oxygen dissociation from haemoglobin is promoted by increasing hydr o ion concentration.H2C0a ionize HCO and H÷ so that the ratio of HCO /H2CO3 becomes 20/1 (substitute this r in Hend erson-Hasselbalch equation along with 6. other words. . (/) It is known that oxygen binding to haemoglobin is weakened by decreasing the pH. the pH of blood l). CARBONIC HC03 +H+ CO2 ÷ 1-12J 1-12J3 How is the H+ generated by incoming CO2 quenched? To answer this question. BLOOD.4 . it comes to 7. The ionization of H2CO8 can cau decrease in pH because it increases the hydrogen ion concentration. have to consider the acid-base properties of haemoglobin. In allwe may that input of CO2 can decrease the pH if H÷ is not eliminated. This phenomenon is known as the Bohr effect after its discove (//) CO2 (diffusing from the tissues into the erythrocytes) does not reside i n the erythroc as such. .4. haemoglobin also contributes importantly to the buffer ing of bl How? Let us consider the following points before we elaborate the whole mechanis m. VII= 7.role of oxygen transport.1 as the pKa for H2CO3 and calct the pH. It is converted by the enzyme carbonic anhydrase to H2COa. the acid form of deoxygenated haemoglo bin (HHb) would be predominant. HHbO (pKg.4 HI-Ib Hb. This automatically means that oxygena ted haemoglobin is a stronger acid as compared to deoxygenated haemoglobin. an amino acid which has a pK of about 6.49 Acids and Bases We have already mentioned that haemoglobin is rich in histidine.62) BLOOD. We can include another H i n this symbol to denote the ionlzable hydrogen. The CO2 in the form of HCO now leaves the erythrocyteS and is carried to lungs t hrough the venous blood. pH = 7. To counterbalance the exit of HCO . . At this point the Bohr effect comes into p lay and oxygen dissociates from haemoglobin and diffuses into the tissues. We have already seen that deoxyhaemoglobin has a higher pK and do es not dissociate to a great extent.4 HbO. of about 8.moves into the erythrocytes from plasma. 7. Thus. This is known as the chloride shift. We denote haemoglobin by the symbol Hb. ).4) converting it to its acid form HHbO2. but in the process the objectiv e of oxygen transport to the tissues has also been achieved. = 6. let us consider the bufferLng of btood. CI. On the contrary at the same pH. while deoxygenated haemoglobin (HHb) has a pK. at pH of blood. This H÷ reacts with the base form of oxygenated haemoglobin.+ H÷ (pK = 8. HbO . Experimental determination proves that oxygenated haemoglobin (HHbO2) has a pK of 6. The acid form of oxy haemoglobin becomes deoxy. pH = 7. (which is predominant at pH 7.18. + H+ BLOOD.18) With these points in background. We have seen that incoming C02 causes an increase in H÷ ion concentration which has to be eliminated . the base form of oxygenated haemoglobin would predominate (HbO .4. What has taken place now i s that not only the H÷ released by incoming CO2 has been buffered. It thus remains as HHb.0 (imidazole group) making it predominantly ionlzable at the blood pH.-62. does not take place because the ionizable hydrogen is quickly donated to HCO to convert it to H2CO3. H2CO3.HbO . 02 binds to HHb converting it to HHbO2. can now indulge in transporting oxygen while it is ca rried by the blood to different tissues.e xits. HCO and 02 enter erythrocytes and CI.HHb02 ge ts converted to HbO .When the venous blood reaches the lungs. by the reverse action of carboni c anhydrase is converted to H20 and . The Bohr effect. however. /I-I2CO3.:-. " :::::::::::! (A} Events in enjthrocyte when arterial blood reaches the respiring tissue.:. and exudates are simil ar t those present in blood. blood contains proteins and m'ay substances possessin phosphate groups.. and Hb-/HHb. CARBONIC : :Z ::.:-'. o :i:i:i:!:i:! .::::::: (B) Events "in enjthrocyte when venous blood reaches the lungs./HHbO. From the foregoing discussion we can conclude that the main buffering systems are the HCO .. Th e systems. Thus. The HCO /H2CO system is much more predominant in these f lui( As compared to this the contribution of potein buffers is much smaller.CO= H+ + HCO= :!:!:!:::: 7. Buffering of blood. Buffers of Tissue Fluids and Tissues The main buffering system present in spinal fluid. Figure I. HbO . 18.:::::::::::: ::::i:? "::::) . lymph.::::::::::i!!ii!!!i! H. Role of HCO / H 2 CO3 and HbO / HHbO2 buffers. . ¢n ::::::::::: i::i::i:i :':':':':':" .:.50 .:::::::::: u I=:: ::::::. In addition. two additional systems which operate in blood are HPO4-/HPO and proteinate-/protein systems. however. contribute less to the buffering of blood than the main system s discusse above. We have already seen citrate in this light. Within the cell many organic acids and their conjugate ba ses also exert buffering actions. Othe r indicator substances also exhibit characteristic change in color with change in pH. The dye exhibits different colors in the dissociated and undissociated states. however. As pH is raised and [H+] decreases. and the pro teinate-/prot¢in. When the dye is half dissociated [HInl=1 Thus [H+] = KI. They are often used for pH measurement when high levels of accuracy are not required (their use has decreased in modern days and the pH meter is the most often used instrument). however. the indicator dye does not dissociate apprec iably. be different from those of litmus.Adds ar Bases 51 In tissues. The indicato r with which we are all familiar is litmus which is red when acidic and blue when basic. What is the mechanism behind such co lor change by indicators? The indicators act like a weak acid dissociating in the following ma nner : Him H÷ + In(acid) (conjugate base) In acid solutions where [H÷] is high. The y show one color above a characteristic pH and another color below that pH or pH range. their colors might. MMMMT OF pH : USE OF INDICATORS Indicators are substances which demonstrate characteristic color properties. is much more here as compared t o their contribution in tissue fluids. . the dissociation of dye becomes predominant til l a stage comes when the dye is predominantly dissociated. We can write an equation for the dissociation constant of indicators. the buffering systems are mainly the HCO /H2CO3. The contribution of the latter. The s ame sample however tests blue for bromothymol blue.J The dissociation constant at equilibrium for an indicator is therefore the value of H÷ at which the color change of dye is half complete. The pH of the given sample. . must be within pH 8 and 9. A negative log of this value wil l give us the pKa of indicator. By using two or more ind icators we determine the pH of a given sample within one to two units. Its pH.9 gives a list of the commonly used indicators. therefore. The pH of the sample must be less than 9. a given sample is colorless for p. therefore: can not be below 8. Table 1.. henolphthalein.LIn. For example. 1 .4 Yellow Red Red Red Blue Yellow Yellow Yellow Bromophenol Blue Bromocresol Green Methyl Red Chlorophenol Red Bromothymol Blue Paranitrophenol Phenol Red Cresol Red Thymol Blue Phenolphthalein .5.52 Table 1.9 .9 Some common Acid-Base Indicators Ind/cator pH Range Acid Color Base Color Methyl Violet Thymol Blue Methyl Yellow Methyl Orange O.2 .4.5 1.8 2.0 3.1.4.2. 6 6.6 8.2 .6.4 .8.6 3.0 .3 4.0 .2 .7.7.8 .Thymolphthalein Alizarin Yellow R 3.5.2 .8 .8 9.0 .8.5 6.3.10.9.9.12.6.4 6.4. 8.1 .0 7.8.5 10.4 4.0 .0 Yellow Yellow Red Yellow Yellow Colorless Yellow Yellow Yellow Colorless Colorless Yellow Blue-Violet Blue Yellow Red Blue Yellow Red Red Blue Red Blue Violet . useless when the test solutions are highly colored or when very high accuracy is needed.Papers impregnated with several dyes are commercially available. These papers are very efficient and can determine the pH of a given sample to about 0. they leave behi nd their loosely bound valence electrons as a negative charge on the electrode. Some of the positively charged metal long might reattach themselv es to the negatively charged electrode. pH meters are used.s to reattach themselves. Electrode Potentials When a strip of metal (electrode) is dipped into water. ELECTROMETRIC DETERMINATION OF pH Earlier on we have seen that indicators are unsatisfactory for measuring pH of s olutions which are colored. The change in t heir colors when dipped into a sample iscompared with a color-pH code on the dispense r. Such situations call for electrometric d etermination of pH. For a student of biochemistry. While the atoms of metal go into solution. 1 of a pH unit. it tends to dissolve owi ng to its solution pressure. For such measurements. P. Its operation is quite simple. indicators are not a good tool for pH measurement. it now attracts the positively charged metal long which have already gone into solution. it consists of a glass electrode which when dipped in to a solution develops an electrical potential depending upon the hydrogen ion concentration a nd this potential is read off the display which is calibrated into a pH scale. These are di scussed below. These papers are. finally becomes balanced and an equilibrium is reached. Let us try to under stand the basic principles underlying the working of a pH meter. Since the electro de has developed a negative charge. The tendency of the atoms to leave the metal and t he tendency of metal ion. however. We have also seen that for works which require absolute accur acy. probably the most familiar instrument is the pH-meter. At . P = Solution pressure of the metal. the potential difference betw een a metal and a solution of one of its salts is given by Nernst equation . they attach themselves to the metal strip increasing the positive charge on the electrode. (//) P < p. 19. This is an altogether different situation because there are many metal long alre ady in solution. the atoms from the metal s trip continue to dissolve as positive long till the accumulated charge is strong enough to opp ose further dissolution.Acids and Bases 53 equilibrium some positively charged metal long remain in solution and the opposi te charge of the electrode and the metal long gves rise to a potential difference.19): P>p P=p "Figure I. Let us now alter the conditions a bit by dipping the metal int( a solution of on e of its salts. Three possibilities arise (F igure 1. (///) P = p. Since the osmotic pessure of metal long in solution is higher. Since the solution pressure is higher. (t) P > p. The solution becomes relatively negative. Taking thermodynamic reasoning into consideration. p = Osmotic pressure of the metal. These metal long will oppose the separation of more metal long from the electrod e and an equilibrium will be achieved early. No potential devel ops. Diagram indicating how electrode potentials arise when a metal st rip is dipped into a solution of one of its salts..The point of'equilibrium in this case will b e dependent upon the relative values of the two opposing forces: the solution pressure (P) of the metal and the osmotic pressure (p) of the metal ions-in solution.. metal long neither leav e the electrode nor the metal long in solution get attached to the electrode. The opposite takes place. The metal strip in this situation acquires a negative charg e as compared to the solution which becomes relatively positive. Since both the opposing forces are equal. . P= solution pressure. T= abs olute temperature. n = valency of metal ion.will be dependent upon n. (1) nF p where E = electrode potential.osmotic pressure of metal long in solution. p. Since osmotic pressure can be regarded as proport ional to concentration. F= faraday (96. We may therefore rewrite equation (1) by replacing the osmotic p ressure term with a term for concentratiort.RT P E = --In-. E . The natural logarithm is denoted by '/n'. Therefore. R = gas constant (8.500 coulombs). We know that RT/F is constant for any given temperature.316joules per degree). the electrode potential will depend solely upon osmotic pressure. P and p. If we use the same metal. the magnitud e and the sign of the electrode potential.. we can say that the electrode potential is a function of concentr ation of the metal long in solution (actually the electrode potential is dependent upon activ ity and not concentration).- . an d p -. of t he solution in which the electrode is dipped. the elec tromotive force of which will be equal to the difference between the two electrode potentials. The electrode(s) which is used as a standard is known as the referenc e e/ectrode.. Since the electrode potential developed by a metal strip is dependent upon the c oncentration of the metal long.{2) where C = concentration of the metal long in solution. is it not possible to measure the concentration of metal long in any given solution by measuring the potential developed by the electrode? Theoretically th e answer is in affirmative. an electric current will pass from one electrode to another.. In other words. We will consider some of the reference electrodes used in electrometric determinati on of pH in the following pages.54 E = RYlnP . E =E1-E2 RT In C2 ( l and P2 cancel out since they 1 = nF C kate identical for the same metal] If we can flx:the potential of the second electrode then the potential of other half-cells can be determined relative to it. The. However. The two electrodes will now develop different potentials. practically it is impossible to measure the potential Of a half cell (one electrode dipping in a solution). If the two electrodes and the two solutio ns are suitably connected. Thus . 1. This problem can be circumvented ff we provide for a second electrode of the same metal dipping into a solution of a different concentration . the problem of measuring the poten tial of a half-cell in the presence of another half-cell is solved by referring all potentials to a standard reference potential. desirable characteristics that a reference . REFERENCE ELECTRODES The basic function of a reference electrode is to maintain a constant electrical potential against which deviations may be measured. 20) and it is arbitrarily assigned a potential of zero under all condi tions. To increase its surface area.5). Various reference electrodes are in use and some of them are discussed below. . This arrangement in toto is known as the standard hydrogen electr ode (Figure 1. The platinum strip saturated with hy drogen gas.electrode should possess are (0 it should be easy to construct. The solution used to provide this hydrogen ion activity is 1. The Hydrogen Electrode The hydrogen electrode consists of a piece of platinum foil dipped in a 1 M solu tion of hydrogen long. and (//) it shou ld develop potentials which are reproducible even if small currents are passed. Hydrogen gs at one atmosphere pressure is bubbled over the platinum foil. the platinum foil ts coated vth platinum black. acts exactly as the metal electrodes described above when dipped in a solution c ontaining hydrogen long.18 M HC1 (the potential produced is dependent upon the activity and not the concentration: see Box 1. is given by e.SN) Fgure 1.30258 = I x 96.315 x T x 2. = Eh.Eho = --log¢ [ H+ ]i Substituting the values of R.Eho If we denote the hydrogen ion concentration in two different solutions in which we have dipped the hydrogen electrodes by [H+]I and [H+]2. the Nemst equation th at we have discussed previously.30258.Hydrogen gas inlet Gas outlet Platinum strip Hydrochloric acid (I.m. If we consider the potential of normal hydrogen electro de as Eho.m. 8. We can measure the pH of an unknown solution if two hydrogen electrodes are imme rsed into two solutions of differing hydrogen ion concentrations. obtaine d on c. the e.ompleting the circuit will be e. and that of the hydrogen electrode in the unknown solution as Eh.20. the [H÷] being consta nt in one of these solutions.500 . Construction of a Hydrogen Electrode. hen by.f.f.m. The electromotive force developed in such a system will be an i ndex of the [H+] in the unknown solution.f.m. Substituting this value we get. n and F in the above equation we have But loge = logo x 2.f.55 lx 96. (volts) = Eh. the e. 500 I°ge 2 Acids and Bases -----. . used exclusively . Due to these disadvantages its use is extremely limited. it is evident that if the potential difference (e. the electrode responds to numerous redox couples and therefore strong oxidants or reductants a re detrimental for pH measurement.Mercury Calomel 56 Biophysical Chemistry We have already seen that the H÷ actity around a standard hydrogen electrode is fi xed at I. the pH of the unknown solution can be easily calculated.f. -. .for calibrating other reference electrodes.log [H÷] = pH. Moreove r. It is. Even trac es of impurities either in the gas or in the solution are enough to poison the electrode. = 0. Since log 1 = 0.00019837 T × pH.log [H÷]2.f. The above equation therefore can be written as 1 e.m.m.f. The Calomel Reference Electrode .m.f. It is also used fo r measuring hydrogen ion concentrations of solutions in which no other electrode will operate satisfa ctorily. The above equation therefore become s e. A source of ultrapure hydrogen is r equired and it is essential to maintain the correct partial pressure within the cell.0. As we have seen in the previous chapter.00019837Tlog I' but log can be written as log 1 .059 From (3). At 25°C e.) between the st andard hydrogen electrode and the electrode in the unknown solution is experimentally determined . we have .m.log [H+]2. Although hydrogen electrode is the standard of reference. however. it is extremely inconv enient to construct and maintain it for everyday work. = 0.059 pH or pH (3) 0. 2 I. 0 .The design of calomel electrode varies for different applications.) is held against the mercury by means of a sintered glass plug or cotton wool. Contact with an outside electrolyte is maintained thr ough a porous ceramic plug.21. The electrode consists of a strip of platinum sealed into glass and allowed to dip into mercury. but a general purpose calomel electrode can be illustrated as in Figure 1. Structural features of a calomel reference electrode . A paste of calomel (Hg2CI2.1 M. The whole electrode is filled with saturated KCI. Side arm for filling KCI Sintered Glass KCI crystals Ceramic plug Figure 1. 059 Acids and Bases The calomel electrode is a lalf-cell and can be represented as Pt I Hg] Hg2C12 I KC1 (saturated) (Every vertical line denotes an interface at which a potential is developed).246 0. a potential of 0.m.0.246 my higher than the situation where both the electrodes used were hydrog en The equation of pH. when a calomel electrode is used as reference would therefor e Calculation of pH (I) From the text we know that at 25°C using saturated calomel electrode pH = (E - . The potential in a calomel hf-cell is derived from the primary reaction H ++ g2 +2e-2Hg The corresponding Nernst equation for this reaction is 0.pH = E .f. the potential which will d evelop be 0. if we connect a calomel and hydrogen electrode together and the electrode is made to dip in a solution of unknown [H÷]. = E° log Hg 2 connects calomel and hydrogen electrodes to a voltmeter. Thus.246 my (at 25°C). If the KC1 solution is kept saturated. All calomel electrodes have a side arm to replenish KC1 solution.059 I e. all the potentials developed in the calom el half-cell will be constant. 0.246)/0.059. When the electrodes are dipped in a solution the voltmeter reads 0.652. What is the pH of the solution ? 0.652 - 0.246 A$. pH = 0.059 = 6.9 pH of the given solution is 6.9 (2) Calculate the potential developed if the pH of a given solution is 2.6. Ans. 0.4 Calomel electrode is extensively used as a reference electrode for pH measuremen t. It is easy to prepare, cheap and quite easy to maintain. The potential developed by this elect rode is reproducible and constant. The Silver/Silver Chloride Electrode Basic design of this electrode is provided by Figure 1.22. It consists of a meta llic silver wire coated with silver chloride and immersed in a saturated potassium chloride solut ion. The potential of this system is derived from the primary reaction Biophysical Chemistry The potential developed may be calculated from the Nernst equation e.m.f. = E°,--l°g[Cl= 0.222-0.0509 log [CI-] (at 25°C1 From the above equation it bec.omes clear that the potential of the reference el ectrode is a function of the chloride ion concentration (activity). In order that the chlorid e ion concentration remains constant at all conditions of humidity, a saturated potassium chloride s olution is used. Electrical contact between the reference electrode and the solution being tested is maintained through a potassium chloride salt bridge. This Junction is made throu gh a porous ceramic membrane embedded in the bottom of the reference electrode. Silver/silver chloride electrode is extremely easy to maintain and it develops a reproducible potential. It is being used as a reference electrode in most of the current Inst ruments. Side arm for filg KCI Saturated KCI -.. electrode KCI crystals -Ceramic plug Figure 1.22. Structural features of an Ag : AgCl reference electrode. 2. THE GLASS ELECTRODE The measurement of pH by glass electrode involves the use of two reference elect rodes, separated by a glass membrane whose function is to establish an electrical poten tial depending upon the hydrogen ion activity of the solution being tested. The design of glass electrodes is enormously variable, but a basic construction is shown in Figure 1.23. It consis ts of a high resistance glass tube with a thin. low resistance glass bulb fused at the bottom . The bulb is - responsible for the pH sensitivity; the rest of the electrode is insensitive t o [H+]. The tube is filled up with 0. I N solution of HCI. Dipping in this solution is a silver/sflverchlor ide electrode. The other reference electrode might be a calomel electrode, but in most current inst ruments this electrode also is an Ag/AgCl electrode. When both electrodes are dipped Into a s ample, the resulting e.m.f, gives the pH of the solution. and Bases Glass stem : AgCl eiectrode J 0. IN HCI -. -- pH sensitive glass membrane Figure 1.23 Diagram of a glass electrode. It is essential to understand the nature of the pH sensitive glass membrane to u nderstand electrode works. X-ray diffraction studies have revealed that the glass membrane consists ia network of silicate and aluminate long (Figure 1.24). The holes in this latti ce-like structure be occupied by cations of varying size. These holes, however, cannot accommodate anions because of the strong repulsion of the oxygen containing long. By careful manipu lations during it is possible to obtain a membrane whose holes can accommodate only H÷ long. membranes will then be sensitive to hydrogen ion activity. ) gen atoms con atoms Catlons Figure 1.24. Structure of a pH-sensitive glass membrane as revealed by X-ray dif fraction studies. How does a glass membrane respond to differences in hy4rogen ion activity?. The belief is that the glass electrode works by an ion exchange process. It is believed that t he membrane consists of three layers, a dry glass layer sandwiched betweentwo hydrated layer s. Let us now see what events can occdr when this glass electrode is placed in aqueous solutio ns of neutral, basic, and acidic reactions (Figure 1.25). We begin with the basic assumption th at in the holes of the lattice-like network of the two hydrated layers, hydrogen long are presen t. (i) When the electrode is placed in a neutral solution : In this situation hydro gen long from the concentrated HCI in the inside of the glass electrode become bound to the in ner hydrated surface. This results in the release of an equal number of protons from the 'hol es' of the outer hydrated surface and the neutrality of the membrane is maintained (Figure I..25 A). 6O H+C" NEUTRAL BASIC CF ' H CF H H+CI" CI' H H+Cl" e cl" H Tj H+Cl" H+ u H+ H Cl" H ." Cl" H CI" H H+CI-Internal solution Biophysical Chemistry ACIDIC -. H+ + OH'Na+----H20 : H+ + OH" Na+-) H20 Cl" H :---b H+ + OH'Na+ H20 H+CI" .- H+ + OH" Na+.b, H20 CI-H =--b H+ + OH" Na+---- H20 H÷Cl" H + + OH'Na +-- H20 H H H+Cl" Internal External External internal External solution solution solution solution Solution (A) (e) (C) Figure 1.25. The glass electrode works by ion exchange mechanism. A schematic di agram showing events at different pHs. go When the electrode is placed in a basic solution : Consider that the basic so lution is that of NaOH which dissociates fully to give Na÷ and OH-ions. Since a high number of OH- long are present outside, the H÷ long from the outer hydrated layer leave t heir 'holes' and go into the sample solution where they combine with OH- long to give H20. The Na÷ lon g remain unquenched and therefore positive charge at the outside of the glass membrane in creases. Let us see what is happening at the inside of the membrane. Since the outside 'holes ' are becoming vacant, more and more protons from the HCI solution inside, become bound to the inner hydrated surface. This leaves the Cl-ions unquenched and consequently the inner side of t he glass membrane develops an excessive negative charge. The separation of charge on two sides of the glass membrane gives rise to the electrode potential which is an index of the pH of the solution (Figure 1.25 B). (iii) When the electrode is placed in an acidic solution : In this situation, th e concentration of H+ long at the outside of the membrane is high. Consequently, the 'holes' in the outer hydrated surface are occupied by H÷ long. Now, to maintain the electroneutrality of the mem brane, H÷ long from the 'holes' of the inner hydrated layer are released. These H+ long de crease the excess of negative charge inside due to CI- long. Thus, while the inside becomes less n egative, the outside is becoming less positive. Again, this separation of charges is the sour ce of the electrode potential (Figure 1.25 C). The overall potential of the glass electrode is a contribution of several potent ials: (/) the potential of the internal silver/silver chloride electrode; (//) the potential d eveloped at the inner glass surface; (No the potential developed due to imperfections in the glass sur face; and (iv) the po.t.ential developed at the outer glass surface. However, potentials (/), gO, a nd (///) are all constant ELECTRODE Reference electrode (Ag]AgCl,or Hg/Hg C12) REFERENCE ELECTRODE Internal referenc electrode Ag/AgCl GLASS Acids and Bases 61 for a given electrode and it is only (iv) which changes with the pH of the test solution. For a given glass membrane, then, all these constant potential may be collected into a gener al constant EG, and an equation may be written. 2.303 RT e.m.f. = Eo + (pH outside) F' We know that E,., R, and F are donstant. Thus, e.m.f, depends only on the pH and the temperature. This equation therefore tells us how important it is to maintain th e temperature of the buffer used to standardize the pH meter and the temperature of the test s olution at the same level for the pH reading to be correct. When a pH sensitive glass electrode and a reference electrode are dipped in a so lution, a galvanic cell is set up. The cell can be written as follows: GLASS CERAMIC MEMBRANE MEMBRANE electrolyte solution (Sat.) (0.1 N HCI) Each vertical line in the above representation denotes an interface and a .poten tial is developed at each interface. The potential of the whole galvanic cell is the alg ebraic sum of the potentials developed by the glass (indicator) and the reference {Ag/AgCI or Hg/H ggCI2) electrodes. e.m.f. (cell) = e.m.f. (re0 3, e.m.f. (glass) -Since e.m.f. (ref) is constant, e.m.f. (ceil) varies only with a variation in th e pH of test solution (if the temperature is constant). The modem glass electrodes develop po tentials which give a linear relationship with pH .changes. J (B) (C} Floure 1.26. Various kinds of pH-s.e.nsttive electrodes. {a) general purpose gla ss and reference electrode: (b) combination electrode for pH measurement of flat moist surfaces; (c) combination micro-spear electrode for small sample volumes. 62 Bophysical Chemistry The glass electrode can be combined with an external reference electrode. In oth er words, the reference electrode can be built in the same unit. Such an electrode known a s a combination electrode is illustrated in Figure 1.26. Also illustrated in the same figure are various.kinds of combination electrodes used for different purposes. The glass electrode can be dsed to measure pH of virtually all kinds of solution s, including those containing strong oxidizing and reducing agents. It may be embedded in sem isolid materials such as cheese or butter and give a satisfactory pH value. It is also suited for pH measurement of biological fluids. It however, has certain disadvantages {which can be circum vented}. Firstly, the electrical .resistance of the glass membrane is high. Use of high impedance amplifiers is therefore mandatory for potential measurement. Secondly, at high pHs (higher tha n 9) the electrode may start developing potentials due to sodium long rather than due to H÷ long. This problem has to be ameliorated by manufacturing a special glass with low sodium c ontent. 3. THE pH METER The pH meter is basically an electronic voltmeter (or potentiometer) designed fo r use with a glass electrode system. It is composed of(/) a reference electrode, (//) a gla ss electrode responsive to the pH of the solution surrounding it, and (rio an electrometer, which is a d evice capable of measuring very small differences in electrical potentials in a circuit of extrem ely high resistance. The pH meters are usually so constructed that the true zero of the voltmeter is at or near pH 7. Before use, the pH meter must be standardized with a pH 7 (or pH near 7) b uffer, adjusting the zero calibration to give the correct reading with the temperature control. Then a second buffer, whose pH is near to that of the sample is substituted and the met er is adjusted to give the correct reading with the temperature control, This procedure helps t o establish the correct linear relationship between my and pH. Moreover, this standardization mu st be done each time a pH measurement is to be done. This is essential because there are su btle changes in various potentials due to the ageing of the electrode. The standardization ju st before a measurement tends to nullify these potential changes due to electrode ageing. To be of use, the meter of pH electrometer must give a reading consonant with th e pH of the sample. This needs the signal to be amplified greatly before it is strong en ough to activate a standard milliammeter or a millivoltmeter. The following equation states that th e pH sensitive, galvanic cell potential (E3 to be amplified is e.m.f. (cell) -- e.m:f. (rei + e.m.f. (glass) But e.m.f. (glass) Therefore 2.303 RT = E +pH 2.303 RT e.m.f. ¢cell = e.m.f.tref +Eo +--pH F or e.m.f. {cell) = E + mpH where E = e.m.f. {rel) + EG, and m = 2.303 RT/F The amplified signal is then directed to a meter which produces a deflection rel ated to the pH of the solution. Acids and Bases 63 In general, the determination of pH using a pH meter is quite simple. The soluti on is placed in a small container. The glass and the reference electrode (or the combi nation electrode) are dipped into this solution so that the glass bulb is dipped completely. The p otentlometer circuit is closed and adjusted till the null point is obtained. The pH can then directly be read off the display. Two general precautions must be taken; (t) As stated above, the pH meter must be standardized before every measurement, and (//) when not in use, the reference a nd the glass electrode must be kept immersed in water so that the pH sensitive membrane does not become dehydrated. The following points must be kept in mind while measuring the pH. Sodium Error General purpose glass electrodes are to a certain extent permeable to sodium lon g also. Thus they can produce a potential for Na÷ in the same way that they do for H÷. There fore in solutions where Na ÷ is present, the actual pH determined by glass electrodes is l ower than what it should be. This is so because the electrode reads a sum of both H÷ and Na÷ long, i.e., the recorded pH is -log |H÷ and Na÷] and not just-log [H÷]. NaturaLly, as the concentratio n of Na÷ increases, the pH decreases. The effect of this problem is not so noticeable whe n the pH is low. But when one is working at high pH (due to NaOH), the pH reading could be off by as much as two units if I M NaOH is used. There aretwo ways to correct this problem. The first is to use Na÷ impermeable gla ss electrodes. However, if this is not possible, then one should work preferably wi th KOH when working at high pH and avoid NaOH if possible. The general purpose glass electro de is not permeable to the bigger K÷ long. Electrode Contamination It is a common precaution to wash electrodes frequently with acid or With deterg ents or sometimes both. The reasons are not fa to see. In our discussion on glass electr odes we have seen that these glass membranes have holes for H÷. Naturally, if these holes are b locked, the electrode Will give a faulty reading owing to reduced permeability. For biochemi sts this permeability problem is quite common since they work With proteins. Proteins can form a thin film on the glass. This impedes permeability. This film can be washed away compl etely with either acid or detergents. Thus, it cannot be emphasized enough that if one is w orking With proteins, the electrode must be washed very frequently with acid or detergents. Certain commonly used buffers react With the components of glass electrodes. One example is that of Tris[tris-(hydroxymethyl)aminomethane] buffer. This buffer can cause large changes in pH readings. One should read the manual supplied along With the electrodes ca refully. The - buffers that can cause changes in the electrode areusually listed there. One s hould then avoid working with these buffers as far as posslble. Concentration of Long Previously we have. discussed that the pH is actually a negative log of H÷ activit y and not H+ concentration. At low concentration the activity and concentration are the sa me, However, at high concentration the activity actually becomes lower than the concentration . Thus, one must avoid measuring pH of very concentrated solution. There is another reason f or doing so. Other long can also affect the activity of hydrogen long. Thus the pH of a buffe r will vary both with its own concentration and with the concentration of other salts in the solu tion: If one is preparing a stock buffer solution which is very concentrated, it is wr ong to adjust the pH of the stock so as to give the correct pH upon dilution.. Rather the conc entration should be so prepared that upon dilution it gives the correct pH; the pH of the diluted buffer should be measured. Biophysical Chemistry pH papers are for rough use. The commercially available pH papers claim that measurements can be very sensitive - difference of 0.2 pH units can be detected. However, there are numerous pitfalls involved here. For biochemists, the pitfalls are hig her, and, again these pitfalls result from having to work with proteins. Many proteins are known to cause errors of several pH units when working with pH papers. High salt concentration can also cause large errors. Additionally, some components of the solution may react with the pigments on the paper and cause color changes not related to pH at all. Suggestions For Further Reading 1, Henderson, L.J., The Fitness of the Environment, Beacon Press, Boston, 1958. 2. Edsall, J.T. and Wyman, J., Biophysical Chemistry, Academic, New York, 1958, Vol. I. 3. West, E.S., Todd, W.R., Mason, H.S. and Van Bruggen, J.T., Textbook ofBi ochemlstry, 4th ed., Macmillan, 1966. 4. Segel, I.H., B/ochem/ca/Ca/cu/at/ons, 2nd ed., Wiley, New York, 1976. 5. Bates, R.G., Determination ofpH ; Theory and Practice, 2nd ed., Wiley, New Yo rk, 1973. 6. Wfllard, H.H., Merritt, L.L., Dean, J.A. and Settle, F.A., Instrumental Methods of Analysis, 6th ed., CBS, New Delhi, 1986. EXERCISE 1. Carbonic acld has two pKa. pK = 6.35 and pK2 = 10.3. (a) Draw a titratio n curve indicating the p, attern of ionization of carbonic acid as a strong base is added. (b) Iden tify the pH ranges where buffering occurs. (c) Identify the predominant chemical species at each of the following pH values : 4,5,6,7,8,9, I0, I 1 and 12. (d) Identify the pH at which HCO specie s would exist in a 50-50 equilibrium with its conjugate base. You are given two buffers : (a) 0.1 M phosphate buffer of pH 7.7 and (b} 0.1 M p hosphate buffer at pH 6.71. If acid is to be added to these buffers, which of them, do yo u think, will resist the pH changes better? When the ratio of salt to acid concentration is unity, the buffer has maximum ef ficiency. Prove this statement mathematically taking an example of acetate buffer, pK of a cetic acid is 4.76. 4. What is the major ionic species present at pH 7.5 in 0.15 Msolution of ( a) leucine, (b) glutamic acid, and (c) arginine. 5. The living cell contains several phosphoester compounds. 3-phosphoglycer aldehyde, whose structure is given below, is Just an example of such compounds. CHO HCOH CH2OPOI2 -, The hydrogen at position 3 are dissociable. The first pKa is 2.1 and the second is 6.8. Can you now draw the structure of the species of the compound that would predominate at physiological conditions7 6. How is it that buffers of different compositions can have the same pH? F or example it is possible to prepare 0.01 M phosphate buffer of pH 7.0 and 0.1 M phosphate buffer of pH 7.0. Acids and Bases 65 7. /k man suffering from untreated diabetes mellitus is admitted to a hospi tal. Doctors fear that his blood pH may have dropped because of ketoacidosis. Analysis of his blood rev eals that [HCO ] = 16 mM and Pco 2 = 30. If pK of HCO is 6. I., determine whether the pati ent runs a risk of acidotic coma. (Note. In plasma under physiologic conditions, concentration of CO2 and Pco2 are related by the solubility constant for CO2 in plasma which is 0.03 mM/mm Hg, 8. After a course of insulin the man is feeling good and his blood pH has become 7.4. [HCO ] has increased to 21 mM. Cm you calculate the concentration of CO2 in the blood p lasma of the patient at this point? Can you also calculate the Pco2? 2 ION SPECIFIC ELECTRODES It is a well known fact that metal long have a profound effect on cellular proce sses. The importance of the role that long play in cellular activity can be gauged by the fact that most cells maintain a very critical Na÷ and K÷ balance between the extracellular and the intrac ellular spaces. Any disturbance in this critical balance is detrimental to the cellular metabolism through a drastic change in the osmotic pressure resulting in cellular swelling. Another metal ion, Caz÷, is known to act as a minatory intracellular messenger stimulating such diverse p rocesses as insulin secretion, chemotaxis, endocytosis, and even cellular proliferation. The se examples are sufficient to underscore the importance of studies evaluating the activity of me tal Long in various metabolic states and processes. The realisation of the importance and increased number of such studies involving long prompted a swift development of glass electrodes so useful for measuring ion act ivity in biological samples. Ion Selective Electrodes Measure the Activity of Metal Long Biological fluids contain many different types of Long such as Ca2+, Na+, IC, CI -, HPO4-, HCO-s, etc. The ensuing electrical interactions between these long will assure t hat every ion, while repelling like charged long, will surround itself with oppositely charged long. Any given ion can therefore exist in two forms in a biological system; (0 it can exist in its free ionized form, or (//1 it can exist complexed with an oppositely charged ion(s), where the net charge of the complex is zero. To understand the point better, let us take an example of calci um. Calcium can exist in its free ionized form. Ca2+, or as a chargeless specie(s} like CaCO 3, CaCI2, etc. It is, however, well known that the effects of calcium, such as nerve conduction, muscu lar contraction, cellular proliferation, etc., all depend upon the free ionlzed calcium, Ca2÷. It i s therefore much better ff the study conducted and the instrument employed measures the concentra tion of free ionized calcium (or the activity of calcium long) rather than measuring the tota l calcium concentration. The same logic applies to the study of other long too. In the lat er pages of this text book, we will discuss about a technique known as flame photometry (emission and absorption flame photometry} which has been employed as an useful tool to measure mineral c ontents in biological samples. This technique, however, measures the total quantity of the mineral present (domplexed or free ionized form}. On the other hand, ion selective electrodes me asure the concentration of the active species of the metal long. Basic Principles An ion selective electrode operates on exactly the same principles as a pH elect rode (see Chapter 1). In fact, a pH electr0de is a type of ion selective electrode sensiti ve to hydrogen long; Just like a pH electrode, the electrode body contains a reference solution and a n internal reference electrode. On to this electrode body is sealed an ion selective membra ne which acts as Ion Specific Electrodes 67 the ion sensor. Four different types of ion selective membranes are in use. They are (i) specially formulated glass, (//) an ion exchanger dispersed inan inert matrix, (/tO a crys tal, and (/v) a liquid ion exchanger. The external reference electrode is either a calomel or a silver/silver chloride electrode. The potential developed across the ion selective electrode c an be measured on a millivolt scale available in a pH meter. This is proportional to the activi ty of ion in the sample. More sophisticated instruments employ specific ion meters (high impedanc e millivoltmeters) which have readout scales directly calibrated in concentration. The presently available ion selective electrodes may be divided into four catego ries depending upon the ion selective membranes that they employ. The four types are discussed in brief in the following pages. Glass Membrane Electrodes The selectivity of a glass electrode is a function of the composition of the gla ss. Three subtypes of glass electrodes and their selectivity characteristics are presented below.: Type : pH, order of selectivity : H÷ >> Na÷ > K÷, Rb÷, Cs÷ . >> Ca2÷ Type : cation sensitive; order of selectivity : H÷ > K÷ > Na÷ > NH, Li÷ >> Ca2÷ Type : sodium sensitive, order of selectivity : Ag+ > H÷ > Na÷ >> K÷. Li÷ .... > > Ca2÷ The second two subtypes are in general responsive to monovalent cations and are more or less unresponsive to anions. Appropriate adjustments ofglass composition Change the degree of electrode selec tivity and also the selectivity order depicted above. Thus, glass can be made more resp onsive to cations by adding to it elements which have coordination numbers greater than th eir oxidation numbers to alkali metal-silicate glasses (20% Na20 - 10% CaO - 70% SiO2). This t reatment excessive negative change to the glass making it suitable to attract cations having a proper charge-size ratio. Glasses with a composition of 27% NaO - 5% Al 2Oo - 68% , show a general cation response. If the above composition is modifiea to 11% Na 2( 18% 7 lO./o SiO, the glass becomes highly sodium selective as compared to other alkali metal . elecfrodes are very sensitive to silver long also, but this does not pose a pr oblem in their biological applications. Electrode stem - Internal reference electrode ljure 2.1. Construction of a typkal glass electrode Glass electrodes are preferred where the studies involve measurement of sodium, lithium, or silver long because of their high specificity for these long. Other desirable feature of the glass is that due to its relative inertriess it can be used in non-aqueous media, orga nic solvents, and Ag/AgCl electrode also in the presence of lipid soluble or surface active molecules. They also sho w an indifference to anions present in the sample unless the anions chemically attack the glass. The glass electrode consists of a stemof non-cation responsive, high resistance glass on which is fused a thin walled bulb of cation responsive glass. Figure 2.1 depicts typical glass electrode. For further details about the working of a glass electrode the reader is advised to refer to the chapter on acids and bases. Solid-State .Ion Exchanger Electrodes In these electrodes, the glass membrane is replaced with a solid-state ionically conducting membrane. The ion responsive material is an insoluble or sparingly soluble salt dispersed in an inert matrix. Often used inert matrices include silicone rubber, polyvinyl chlor ide, and other polymeric materials. In order to prepare the membrane, the ion exchange material is dispersed through the inert matrix. Such membranes have good mechanical properties and giv e reproducible potentials. The membrane so prepared is then cemented to a glass or epoxy resin electrode body. The body holds an internal-reference solution and a reference el ectrode. Sometimes, the back Of an ion responsive membrane is coated with mercury, and a platinum wire is connected to it which works as the reference electrode. These electrodes are mainly anion responsive and several varieties are available for measuring the halide ion concentrations. The ion responsive material for such ha lide response electrodes is usually a silver halide. Reference halide solution Membrane (Silicone rubber wiib silver halide) Figure 2.2. Diagrammatic representation of a solid /on exchanger electrode. Solid-state ion exchanger electrodes have two drawbacks. The first drawback is that they have a relatively short working life. This problem has been circumvented by building the sensor membrane into a removable cap which can be replaced as required. The second drawback concerns itself with the extremely high resistance of silicone rubber and other matrices. In order to tackle this problem it is necessary that the' embedded ion exchange material should provide enough electrical conductivity across the membrane. This is achieved by careful dispersion of the ion exchanger so that each exchanger particle is in contact with each other within the matrix. Figure 2.2 is a diagrammatic representation of a solid-state ion exchanger electrode. Solid-State Crystal Electrodes A crystal or pressed pellets of an insoluble salt can act as ion sensiti ve elements operating in much the same way as the salt dispersed in an inert matrix. The cryst al is precision ground into a disc shape and fixed onto an electrode body. The manufacturing pr ocess is closely controlled to avoid the crystal developing an internal crack or leak. The crystal s hould also not have high resistance. Examples can be cited of the lanthanum fluoride electrode (m easure fluoride) and silver chloride electrode (measures chloride). A single crystal of LaF3 acts as the sensing membrane in a fluoride elec trode. However, LaF3 has a very high electrical resistance. To cancel this detrimental p roperty LaF- crystal is ' 3 usudly doped .with europium (II) which lowers the crystal resistance and facilit ates ionic charge transport. The LaF3 crystal, sealed into the end of a rigid plastic tube, is in contact with the internal solution arid the external solution. The internal solution is 0.1 M wit h respect to NaF For example. as described for fluoride electrode above.lanthanum fluoride membrane. A mobile ion adjacent to the vacancy defect moves into the vacancy. Conductance in the crystal is a function of lattice defect. Thus only the mobile ion takes part in conductance.M. Figure 2. if used at high temperatures. In a similar manner. The electrode thus responds only to fluoride long. This fluoride electrode c an measure the fluoride ion activity as less as 10. bromide. NaCI is present in the internal solution so that the chloride ion can fix the potential of the internal Ag/AgCI reference electrode. size and shape of the particular ion. polycrystalline Ag2S membrane gives a good sulphide ion electrode. Cross sectional view of a crystal electrode. this does not pose a problem as hydroxide ion concentration is kept constant with a buffer. all other long being rejected by th crystal membrane.3 is a diagrammatic representation of the crystal electrode. and thiocyanate. Liquid-Membrane Electrodes .Ion Specific Electrodes 69 and NaCI. Only the mobile ion may move into this vacancy since this vacancy is tailor made for the charge. iodide. The fluoride ion activity controls the potential of the inner surface of the. their life gets shortened considerably (1-3 months). in a fluoride electrode only fluoride long can move into the vacancy defect thereby taking part in conductance. Solid-state crystal electrodes have a life of about 1-2 years. However.3. The fluoride ion may also rspond to hydr oxide ion concentration. Mixed crystals of AgX-Ag2S compose the anion selective electrodes for chloride. Reference electrode Internal filling solution Crystal Figure 2. However. This type of electrod es are used for the measurement of calcium. also known as the membrane. The pores of the disc are roughly 100 rn in diameter. The porous disc. Reference electrode Ion-exchange reservoir Internal filling solution (Reference solution) Porous membrane Figure 2.4). The design of such electrodes is discussed briefly. This process allows change in the ion se lectivity of the electrode. nitrate. Thus. The outer tube contains the ion exchange solution wh ich saturates the pores of a hydrophobic porous disc. is replaceable. Cross section of a liquid ion ehange electrode. They have a typically double concentric tube arrangement in which the inner tube contains the aqueous reference solution and the internal refence electrode. Another type of liquid ion exchan ger used for . Most available electrodes use a porous diaphragm (glass or ceramic disc) which separates the inner ion exchanger solution from the test solution. the ion excha nge solution can be changed after removing the disc. T he ion exchange solution keeps the disc always saturated (see Figure 2. The liquid ion exchanger used for calcium sensitivity is the calcium salt of his (2-ethylhexyl) phosphori c acid (d2EHP) dissolved in various straight ch. perchlorate and other long.ain alcohols. selectivity being a function of the ion exchange solution.The sensing element of these electrodes is a layer of organic solvent in which a n ion exchanger is dissolved.4. The crystal electrode consequently loses its sensitivity to the desired ion. Thus. Interferences Two main types of interferences are encountered by ion selective electrodes. Surface reactions between long present In the s ample with one of the components of the crystal can lead to formation of a second Insoluble comple x on the crystal. high barium ion concentration In a samp le beIng measured for calcium can be detrimental to the experiment as barium long can com pete with calcium long for passage Into the membrane. or all of such factors as the pH. Another type of electrode Interferen ce occurs chiefly with crystal electrodes.s are mostly due to the passage of an Interfering ion Int o the membrane In lieu of the Ion being measured.70 BoplujscoJ Cherrtstrtj calcium is didecylphosphorie acid dissolved In di-n-octylphenyl phosphonate. and (tO electrode Interference. The method Interference can therefore be done nditions diligently.months). 10-phenanthrolIne Ion exch anger which ts sensitive to nitrate. Sometimes one of the component s of a crystal may form a stable cbmplex with anion In the sample being measured. Due to the loss of ton exchanger with each measurement. At the end of such periods. a low pH might give very low values so sInce fluoride long form complexes with the hydrogen long and ivity. Thus. while me for the activity of this ion. ioni are not properly fixed. The electrode Is sensitive enough to measure f airly low concentration of tons. . Method Interference can occur when either c strength or temperature of the sample beIng tested asuring fluoride long. life of such electrodes Is extremely short (1-3 . are (0 method interference. This is consequently lose their measurable act away with by fixing the measurement co Electrode interference. Thi s calcium electrode can be changed Into a nitrate electrode ff the calcium sensitive Ion e xchanger In the electrode Is replaced by a substituted nickel (II}-1. the ion exchanger Is refilled a nd the porous membrane replaced with a new one. Example may be cited of a bromide electrode (AgBr crystal) which forms a complex with thiocy anate ion (AgSCN) altering its sensitivity to bromide long. For example. The y. Thus. extent of precipitation. degree of acidity. This results In Increased solubi lity of the membrane thereby IncreasIng the lower limit of detection of the fluoride ion. solution conductivities.use falls In the area of dental research. the chloride electrode is beIng used to assay chloride ion activity In the sweat of babies as a diagnostic test for cystic fibrosis. (/0 Ion selective electrodes can have many diagnostic applications In biolog y. (/to Ion selective electrodes are used to study the control of ion transport b-y tissues and cells. For example. Example can be cited of very recent studies by Prentl¢l eLa/where Ca2+ home ostasis of human neutrophlls was studied under various stimulating and resting condition s. The se are some of the Industrial uses of ion selective electrodes. . formation of comple xes. Calcium and fluoride electrode s are beIng used to study the relationship between tooth decay and saliva ion composit ion. Transport of Na ÷ and C1. Another application with a potential f or direct health . ion electrodes have been used for predi cting corrosion rates. and effectiveness of electroplating bath solutions. Such studies have provided a deep understanding of the role of long In triggerin g neutrophil response to an InvadIng antigen.long have al so been studied In various ceils usIng the correspondIng electrodes.citrate long form a very stable complex with lanthanum long. Applications (0 Activity measurements are valuable because the activities of Long determ ine rates of reactions and chemical equilibria. ."'X I I Ion-selective f" -----.----'-'. is converted into a urease sensitive electrode and can measure the activity of urease in various samples.10-I . For example. There are thousands of other enzyme . the ammonium ion electrode can be converted into an enzyme electrode ' measuring the activity of urease. Re fe r en c e solution Ge.--. The enzyme urease (kO present in the sample will then act upon urea in the membrane to give rise to ammonium long which can diffuse through the membrane to be sensed by the electrode. The ammonium electrode thus. Urea is fixed in a gel membrane which is fastened onto the bulb of ammonium electrode.-" membrane t--.--. i .100 .100 .----..I0° Chloride Cupric Cyanide Iodide Nitrate Nitrite Sodium Ion SpecIj Electrodes 71 (/v) Another very stimulating biological application of ion selective electrodes is that they can be converted into enzyme electrodes."M .10-2 . These electrodes can then measure the a ctivity of an enzyme in a given sample [the only action required on the part of the investigator is to dip the electrode in the desired sample).ob ed subtrate or enzyme 100 100 10-2 100 . volatile amines S2-. to this tremendous utility potential. 2.5. As is obvious from some of the applications listed above.5 .I0° i0-s10-7 10-6 -10-7 lO-5 lO-6 lO-6 Potassium 10-5 Lead 10-7 Range (M} Interfering Long and Compounds CO2.1. Construction of an enzyme With ion selective electrodes. ion selective electrodes can provide much useful information about cellul ar processes. The design of an enzyme e/ectrode. I- . I Particulars of Some lon Selective electrodes Material Detected Concentration Ammonium 10-6100 Bromide 10.5.substrate combinations that yield products measurable Figure 2.100 Calcium I0-5. a lot of ion selective electrodes having s ensitivity for long have been developed in recent years. A list of such electrodes along With t heir and the long which can interfere in their functions is provided in Table 2. electrode is provided in Figure 2. ICO K÷. the carbon dioxide electrode is chiefly used to measure carbon dioxide in the bl ood. the oxygen electrode and the carb on dioxide electrode. Li÷ H÷. Mg2+ I-. NO. Cu . GAS-SENSING ELECTRODES Two gas electrodes are Widely used in biology. HCO. Br-. Fe2+. NH Ag÷.riefly in the following pages.Zn2+. Both of these electrodes are described b. pb2+. S2-. V S2NO. SO-. . Hg2+. F-. Whereas the oxygen electrode is used in many diverse branches of biology. Br-. Ag2+ S2-. Cs÷. These electrons are sup plied by the platinum cathode. (//) The outermost valency shell of each oxygen atom has a vacancy for two el ectrons upon acceptance of which it can be turned into an oxygen ion.6 V is achieved with the help of a mercury cell. The reaction in the electrolyte is : OH. Polarization of elect rodes at 0. The gas electrodes. The reaction at the anode is : .72 Biophysical Chemistnj The Oxygen Electrode Although a variety of different anode-cathode combinations for oxygen electrode are available. The electrodes dip into an electrolyte sol ution (usually a buffered pgtassium chloride solution) which is held inside an electrode by an ox ygen permeable membrane. When the oxygen electrode is dipped into a solut/on containing oxygen.+ KCI -. the platinum with silver/silver chloride is the most used cathode-ano de combination. including the oxygen electrodes measure the partial pressure of a gas in solution rather than its concentration. The often found arrangement of these electrodes is annular with the tubular silver/silver chloride anode enclosing the platinum cathode. where X stands for the gas whose partial pressure is being indicated. the follo wing reactions happen : (i) The oxygen molecules from the sample diffuse through the membrane into t he electrolyte so that within a short time the electrolyte and the sample come to equilibrium w ith respect to the pO2. The membrane might be a very thin polypropylene.KOH + CI(iv) The negatively charged chloride ion produced in the electrolyte solution are attracted to the positive anode and donate their electrons. The term partial pressure is often ab breviated to pX. the partial pressure of oxygen is indicated as pO2. and that of carbon dioxide as pCO2. Thus. The reaction at the cathode is : 4e-+ 02 + 2H20 = 4OH(//0 The hydroxyl long so produced at the cathode then react with potassium c hloride in the electrolyte solution. = C1 + eAg + Cl = AgCl There is thus a deposition of silver chloride on to the anode. of the sample.01 M borax. If the oxygen concentration remains constant. If this can not be done. Calibration of the oxygen electrode is done with the help of an oxygen free samp le (5 mg sodium sulphite in 5 ml 0. . The overall result of the above reactions is a transfer of electrons from the ca thode to anode. It is thus imperative that the sample measurement shou ld be done at the same temperature at which the calibration was performed. a temperature variation of IC would induc e a 5% variation in the current.CI. or water through which nitrogen gas has be en bubbled) and another sample with a known pO2 (equilibrating a liquid with air with a know n oxygen content). This transfer represents a current flow which can be measured and is prop ortional to pO2 of the sample. The current produced by an oxygen electrode is affected by variation in temperat ure. The response of the calibrated-electrode is exactly linear to the part ial pressure in the ample. then temperature correction must be applied before one reaches to a conclusion about pO2. Electrode body Platinum cathode Ag/AgCI anode Membrane Electrolyte Figure 2. usually 0. The same temperature variation relationship di scussed with respect to the oxygen electrode applies here too.6. . Construction of an oxygen electrode. D/agram of/t carbon d/ox/de e/ectrode. (/0 a thin plastic or teflon membrane which is permeable to carbon dioxide and not to other long. This pH change is reflected by the pH meter which is directly calibrated for pCO2.7 . When the electrode is dipped into a sample containing dissolved carbon dioxide 0 the carbon dioxide is allowed to diffuse into the bicarbonate solution by the permea ble membrane.7. and this change is read by the glass electrode. The Carbon Dioxide Electrode The constituents of a carbon dioxide electrode are (/) a conventional glass pH e lectrode with a calomel reference electrode. A diagram of the conventional carbon dioxide electrode is provided in Figure 2. Th e response time of a carbon dioxide electrode is higher because the standard bicarbonate solutio n has to come Into equilibrium with the sample.73 Ion Specific Electrodes A diagram of a typical oxygen electrode is provided in Figure 2.005 M NaHCO3 between the membrane and the glass electrode. The pH of the bicarbonate solution changes.6. and (///) a standard bicarbonate solution. Combined pH and calomel reference electrode Standard bicarbonate solution Membrane F/gure 2. .n for measuring the carbon dioxide dissolved i n blood or plasma. oft.e.Applications The oXygen electrode is being widely used in many different biological experimen ts wherever there is a need of measuring oxygen. The carbon dioxide electrode on the other h and is mainly used for clinical purposes. 74 Biophysical Chemistry Suggestions For Further Reading I. lon Selecave Electrodes. No. New Delhi. N. LL. 1976. Dean.. F. Membrane Electrodes. Willard. Cambridge University. CBS.. . Instrumental Me thods of Analysis. New York. Merrltt. 1986. J. Lakshmlnarayanaiah.. Cambridge monographs in Physical Ch emistry. and Settle. J. 2.H. 1975.A. Koryta. 6th ed. Academic. 2.. H. 3.A. New York.. The breakthrough in colloidal chemistry came with the work of Thomas Graham. All protoplasm i8 in colloidal form. He observed that solutions of certain substances diffused at a slower r ate and were unable to pass through a parchment membrane. sulphur. Historical Perspective The study of colloids as a system began with Selml in 1843 when he prepared coll oidal suspensions of Prussian blue.3 THE COLLOIDAL PHENOMENA Until the middle of the nineteenth century colloidal systems were regarded as be ing outside the realm of well behaved chemical systems because they did not behave in a mann er expected of an aqueous solution. milk. notably blood. On passing a narrow beam of light through this preparation he observed the path marked out by a cloudy haze. ele vation of boiling point. He observed that the preparat ions were not true solutions but suspensions of a finely divided state of matter in water. colloids must constitute extremely well behaved systems because life is a manifestation of various colloidal states . lymph. This primitive system of classificatio n was not fortunate as it was seen later that many substances which formed glue-like solutions could . bile. electrolytic conductance. lowering of vapour pressure. which is due to diffraction or scatter ing of light by colloidal particles was further studied in 1969 by Tyndall and is today known as the Tyndall effect. The above phenomenon. depression of freezing point etc. Because of their sticky nature he s upposed them to be noncrystallizable and called them co//o/ds (Kolla in Greek means glue ). Substances belonging to the other class which were readily diffusible through membranes and also could be crystallized were termed crystalloids. Most of the biological fluids. prepared aqueous suspensions of gold in 18 57 and studied the optical properties of this preparation. Moreover. This could no t be observed in a true solution. However. Such physico-chemical properties of colloidal solutions as the exhibition of osmotic. an Englishman who performed certain fundamental experiments to prove the existence of colloidal systems. and digestive secretions are colloidal solutions. Michael Faraday. the biomembranes may themselves be considered to be a manifestation of the colloidal state. pressure. a British scientist. were different. and casein. The appearance of a colloidal solution under an ultramicro scope is like a milky haze of counfless points too minute to be separately observed. Only when these two phases i. According to them. a rough classification was suggested by Siedentopf & Zsigmondy. be brought into a state of subdivision to be appropriately termed colloidal.e. the dispersion medium.be crystallized. Thus a colloidal system is a state of matter and not a type of matter. On the other hand from almost all the crystalloids colloidal solutions could be obtained. With the invention of the ultramicroscope in 1903 the study of the properties of colloidal sols became possible. the particles which were distinct in the ordinary microscope . the system can be said to be colloidal in natur e. Wolfganf Ostwa ld in 1907 pointed out that any matter in finely divided dispersed condition implies the presence o f another phase. the dispersed phase and the dispersion medium exist as separate phases.. It was soon recognized by Von Weiman that any substance can under suitable conditions. Based on th e various size -of partlcles which could be distinguished. Hardy added to th is information by looking at it from a quantitative angle. (see Figure 3. T hose wltch could be observed only in the ultramicroscope were the submcrons and the others which were invisible both in ordinary and ultramicroscope were the am/crons.was introduced into medicine as a diagnostic aid to obtain information about certain types of diseases.4% NaCI 76 Boptu3sc Chemlstrg and appeared as well focussed images in We u]tramJcroscope were called mcrons. Zsigmondy introduced the term gold number while explai ning the protective action of one colloid over another. the gold number of cerebro spinal fluids. Co|omNumber of sol 5 Colourless 4 Pale blue 3 Blue 2 /Lilac/purple Red-blue 0 A = Neurosyphilis B = Meningitis to . such as meningitis. Later.Brilliant red-orange Spinal fluid dilutions with 0. neurosyphilis etc. The stability of colloidal systems was studied at great length.1). one of them being clarification of drinking wa ter by adding small amounts of calcium salts. formulated as "the Schu ltz-Hardy rule" are still in practical use today. The importance o f valency and its influence on stability of sols was studied by Schultz. Their findings. They can be formed either by aggregation of small molecules like sodium chloride or by disintegration of large polymers. notably Freundlich {phenomenon of adsorption). is uniformly distributed in a continuous medium. This unpredictable chao tic movement . A colloidal system is characterized by particles ranging in size from 1 ml to 0. Donnan (Donnan equilibrium) Svedberg (sedimentation) and Einstein. finely divided. nor so small that they can be s aid to be in solution. However. The particles are non-filterable and can be observed under the ultramicroscope as illuminated discs engaged in a kind of random zig-zag mot ion. the more rapid is their movement. Gold sol curves for cerebrospinal Jluld give djnostic information Outstanding contributions in the field of colloidal chemistry have been made by many other scientists. This means that the colloidal state is an intermediate state between a suspension and a true solution. The matt er. colloids represent a state of subdivision of matter. the dispersed particles are neither so large that they separate on standing. I. This type of movement is called Brownlan movement after the name of its discoverer Robert Brown. 1 in diameter. The smaller the particles.Normal spinal fluid lgure 3. What are Colloids? As stated earlier. But ff the room is now darkened and a concentrated beam of light al lowed to enter shutter.2. we can see the dust particles in the path of the light clearly. An ord inary microscope is enough to observe the participants of the dispersed phase in a suspension. Only an electron microscope can be used for this purpose as the size of the particles in such a system is always less than I m.The Colloidal Phenomena 77 of molecules was explained by Albert Einstein in 1905. Consider a room with a small shutter on on e of its walls. In that case. (B) lJht beam is scattered and appears as a white path through a colloidal solution of blue ink i n water .2). These colli sions impart sufficient kinetic energy to the colloidal particles so as to enunciat a Brownia n movement. Tyndall effect. Minute dust particles affect the visible light wa ves to produce the phenomenon called diffraction. (A) No licjht scattering by a concentrated solution of copper sulphate. the two phases separate on standing and the particles can be filtered off easily. however considerably slower than that of the molecules of the medium and can therefore be observed under an ultramicroscope. This movement is. As long as the room is uniformly illuminated the air inside the room seems perfectl y clear and transparent. If a narrowly defined concentrated beam of light is allowed to pass through a colloid al system it appears as a white path. True solutions do not respond to such a phenomenon because its particles are too smal l to scatter (A) Copper sulphate solution ": (B) Colloidal solution of blue ink n water. The surrounding molecules of the dispersion medium continuously collide with the colloidal particles. The same is not observed in a true solution (see Figure 3. In contrast are the true solutions whose individual members are invisible even in the ultramicro scope. This is due to '. Another characteristic property exhibited by colloidal system is the Tyndall eff ect. If the particles cross the upper size limit they will form suspensions. Fre 3. The same principle is followed by colloidal p articles. The above effect can be illustrated as follows. of light by dust particles. 3). The images though not visible c an be recorded photographically. Modern methods have made the use of u ltraviolet light to extend the range of microscopic work. Other light is excluded from the field.ndall effect is utilized in the ultramicroscope. (A) Colloidal panicles appear as illuminated discs of light (B) Particles of true solution an: not visible " " . short wavelengths permit for mation of focussed images of objects as small as I0 mm in diameter. Figure 3.4 shows a simplified version of an ultramicroscope. The individual sol particles scatter light and appea r as bright discs gainst a dark background (see Figure 3. View under ultmmlcroscope.beam from an arc lamp is focussed by the lenses of a compound microscope on the stage of another high-power microscope with its optical axis at right angles to the first one. In this instrument an in tense light .The "I. Its. 1) and it is difficult to classify colloids strictly. also called the discontinuous or internal phase is made up of colloidal p articles while the dispersion medium. colloidal systems are composed of two phase of matter.1 Typee of Colloidal 8ytem DiIrd Diponslon Medium Type Solid Solid . various attempts have been made and the classification of colloidal systems as two distinct types the lyophilic colloids and the lyophobic colloids. The dispersed phase. However. Numerous types of combinations of these two ph ases are possible (Table 3. Tyndallization and observation in an ultramicroscope CLASSIFICATION OF COLLOIDS As mentioned earlier. also called the continuous or extema/phase is made up of the solvent in which the dispersion takes place. Table :S.4.78 Solid Liquid Sol Bophysal Chemtry Hlgh-power microscope " 'i Scattered light Source o O microscope Colloidal solution O O Figure 3. based upon the interaction of phases has fdund wide acceptance. Solid sol Solid Liquid Liquid Gas Solid Liquid Solid aerosols Solid emulsion Emulsion Liquid Gas Gas Gas Gas Solid Liquid Gas Liquid aerosol Solid foam Liquid foam . Smoke. water in oil. dust in air. froth on beer.Alloys. volcanic dust. clouds. dispersed metals. glasses coloured with. . Milk. cheese.. cream. ruby glass. Whipped cream. Not known (this type is not seen because of the high rate of diffusion of gases). gemsl pearls. off in water. mist. Butter. Pumice stone. Gold sol. ferrichydroxide sol. Fogs. starch in water.g. e. When it has water as the continuous phase it is called a hydr osol. Also.They are stable and therefore are pr ecipitated only by high concentration of electrolytes. Other significant compound s are the cholic acids. To explain this further. soaps. the great disparity in size of the solute particles confer upon them s ome distinct physical properties. Additional properties are conferred upon them because of th eir charged polyelectrolyte nature. the precipi tation or flocculatlon of these systems is difficult. One unique property of colloidal solution is the Donnan effect.. nucleic acids and the starches fall in this category. The emulsoids are easy to prepare and though they resemble the true solutiohs in many respects. Lyophilic colloi dal solutions differ from true solutions only with rspect to the size of the particles which l eads to a change both in the properties as well as techniques of study of these systems. Numerous substances of biological interest such a s proteins. It is unequal distribution of diffusible long across a semipermeable membrane due to t he concentration of colloidal particles on one side of the membrane. . alcosol. But the general term for this type of system is the emulsoid. they exist in a state of true reversib le equilibrium. It has also been found that the viscosity ihcreases with"concentration. Due to the high degree of solvation of the particles in an emulsoid. and the emulsifying agents. the dispersed phase of lyophilic colloids when separated b y evaporation or precipitation clump together in loosely packed aggregates. Needless to say that the these are colloidal systems where there is strong attra ction between the solvent and the particles.The Colloidal Phenomena 79 Lyophilic Colloids The word lyophilic stems from the Greek word philos meaning loving (lyo means so lvent). Two reasons are attribute d to these observations. synthetic detergents. while With alcohol as its dispersion medium it is called an. Theseflocs can be rec onstituted into the colloidal form easily by removing the electrolyte from the medium and by mix ing: The hydrophillc colloids show a higher viscosity than that of the medium. A lyophilic colloidal system can take up different names depending on the nature of the dispersion medium. No wetting and theref ore no dispersion was poible. .o prominent. If the solute molecule completely repelled the dispersion medium it would have remained dry. lyophobic systems do form dispersed systems in w hich the molecules remain detached from the solvent though not in complete isolation. the difference in refractive indi ces between the dispersed phase and the dispersion medium also affect tla In emuloid this differ ence is not appreciable because of the high solvation character of this system. Therefore the Tyndall effect is not s. This extensiv e solvation of the particle immobilizes the bound water and resists its freedom of movement thu s . there is more trapping of solvent. Also the colloidal particles try to orient themselves in a lattice like structure.(/) The hydrophflic colloids have strong attraction for water. Tyndall effect for these systems is minimum. (//) With higher concentration of solute. This systematic arrangement affects the flow of the solvent as the solvent now has to find gaps in this stru cture to escape or it has to break down this structflre to enable its flow. The lyophobic colloids derive their name from the Greek word phobe (meaning fear ing or hating) and thus mean 'solvent-hating'. But we carmot call these ystems 'solvent hating' in the strict sense because if it was so. However. -. Thi s is one of the reasons of. The optical properties of lyophilic systems are also different. Besides the size of the particles. no dispersion would be formed. increasing viscosity.- .their instability. This electrical potential difference arises due to the charge carried by the particles of a sol. workers were doubtful wheth er the laws of thermodynamics would be obeyed.80 Biophysical Chemistry The lyophobic systems are also called suspensoids. ( Size: A feature of solutions of macromolecules is that the solute molecule is many times . only the scale is different. Since diffusion was very slow. (ii) the bon ding interactions and frequently charged nature of the solute. We now know that colloids do conform to certa in fundamental physico-chemical laws as systems containing only. viscosity was high and fr eezing-polnt depression was not so remarkable in these solutions. The viscosity. These systems are characterized by solute molecules which stay apart from each other. The two factors which determine the solution behavi0ur of biopolymers are (1) th e great difference in size of the molecules of the solute with the solvent.-ndall effect is distinct and therefore the particles of a suspe nsoid are more clearly visible in an ultramicroscope. Research has shown that it is t he electrical potential difference between the surface and the solution far away from the part icles that determirtes the diuturnity (stability) of the lyophobic system. and the charge carri ed by the long in the surrounding medium. The. small molecules or long. To understand this. though.1). is not so affected as the solvent flow is not hindered. despite their intrin sic instability they remain unchanged for long periods (see Box 3. let us first look into the factors w hich are the sources of non-ideality of colloidal solutions. The optical properties of lyophobic systems are worth mentioning. They easily precipitate out f orming irreversible flocculates. This allows increased scattering of light as the difference in refractive indices of the solvent and s olute particles is quite high. But if they are left Undisturbed. BASIC THERMODYNAM/CS OF COLLOIDAL SYSTEMS In the early 1900's when the physico-chemical behaviour of solutions was being s tudied there was a misconception that the basic principles of physical chemistry were n ot applicable to colloidal solutions. The departure from ideality thus depends on this molec ular xcluded . the term entropy is used to denote such a randomness in a system. According to the second law of thermodynam ics. One of the determinants of the equilibrium state of a system of many particles i s the randomness or disorder of the system. In these solutions th e centre of each molecule is excluded from a volume of solution because it is temporarily occupie d by some part of another macromolecule..larger in size than the solvent molecule i.e. In these solutions the centre of each molecule is excluded f rom a volume of solution because of the high solute to solvent size ratio. the solutions are more concentrat ed in terms of weight or volume percent while their molarity is still very low. But this cannot hold true for macromolecular solutions because of the high solute to solvent size ratio. In an ideal soluti on it is assumed that the solute molecules are of the same order of size as the solvent molecules so that the distribution of solute molecules in the solvent is entirely random as is seen in the case of true solutions. . NO B.. 8Mv For spheres.. The excluded volume is determined.2M2 where N is the Avagadro's number... (3) ... (I) . N 4v so that B M 2LMv and for rods. The quantity B. 6 Nd Lv so that B dM .by the actual molecular dimensions. (4) . (2) ... The Colloidal Phenomena 1 volume (6). and M is the mo!ecular weight... the second virial coefficient. which serves as a con venient measure of solution non-ideality is related to '#' by the equation. F= &H -T&S . This liberation of energy is the driving force for mixing. (6) . The globular macromolecules contribute less to non-idcality of a solution than t he asymmetric rods or random coils because the permutations and combination of arra ngements possible in a solutiol for the latter are always less in number than the former. in an ideal solution. (A) The internal energy = sum of internal energies of its components in thei r standard states. (ii) The bonding interactions : When a colloidal dispersion is prepared. the sol ute-solute i Interactions and the solvent-solvent interactions are broken with utilization of energy and new bonding nteractions. AV or volume change for mixing is zero.. (C) On mixing. If the temperature and pressure are as sumed to be constant. and (B) The volume = sum of the volume of the components is their standard state s. E or energy change between system and surroundings is zero. the change in enthalpy (heat content) is also zero. d is the diameter of the rod. i. If we do not consider the 'entropy' term described earlier. When the volume change is nil at constant p ressure. The description of thestate of a solution needs to specify the amount of compone nts present and the temperature and pressure. L is the rod length.. dissolution of a solute in a given solvent will be favoured o nly if the solventsolute interactions are great enough to overcome the solute-solute interactions and the solventsolvent interactions.e.v is the specific volume. The combination of the statement of the first and second laws of thermodynamics gives us the equation. . i. there is a great disorder so that AS or change in entropy is posi tive.e. the solute-solvent interactions are formed with release of energy. . Since it is the free energy of a solution which is a property of interest. On the other hand. for mixing is positive. In ' poor' solvents. The total free energy Of the solution is then F = Z nl]i . Otherwise there will be further reaction or migration of matter from a region of higher chemical potenti al to a region of low chemical potential... there is preferential attractio n between the solvent and solute molecules so that the solute molecule tends to extend itself and the entropy inc reases. the reaction will proceed spontaneously t o form an idea] solutlon. The partial molar energy is the amount of free energy contributed by each component of the system. symbolized by ''. the chemical potential of the solvent is import ant as many macromolecules interact strongly with the solvent or with one another. The virial coefficient win be negative. It is an intensive variable and is often called the chemical potential. the system is sai d to have attained equilibrium. When the value of AF = 0. In that state.82 Biophysical Chemistry It follows from the above equation that if AS. Both the above cases are deviati ons from ideallty. It also follows that tl must be identical at all places in the system. It also determines the direction in which the net reaction will proceed. Only under these conditions. (7) i=] where ni is the number of moles of component L The chemical potential is the int ensity factor of a chemical reaction and may be regarded as its driving force. & F for mixing will be negative. This increases the solvent chemica l potential and enthalpy change then is not equal to zero (AH 0). we mu st know the contribution of each component of a system to the total free energy. For solutions of macromolecules. in 'good' solvents. The excluded volume is larger in such solvents and the virial coefficient is positiv e because there is a great decrease in solvent chemical potential. the solute-solute interactions are preferred. the chemical potential of any com .ponent m ust be constant. At higher temperature s gels generally liquefy. Nageli described a mesh lik e structure for gels. This is attributed to the electrical charge carried by the colloidal system. On the other hand. The second virial coefficient.There. Also. then becomes nll and the solution behaves ideally. B. Many factors li ke pH. are of course. gelatlon is favoured. many of the lyophobic colloidal systems persist indefinitely. Change in H÷ concentration can bring about a change in the molecular constitution of a colloidal particle thus altering its power to gelate. With increasing concentration of the co lloidal particle. When a warm concentrated solution of starch is cooled. A detailed discussion of the electrical properties of colloids will be d ealt with elsewhere in this chapter. This product is called a gel and the process by which it is for med is termed gelation. temperature and. the gel structure is more firm with higher concentra tion of solute. The lyophilic colloids mostly behave as true solutions. To make a solution behave as an ideal one. a temperature can be found (0 temperature or Flory temperature) at which the above enthalpic and entropic t erms cancel each other. concentration of solute as well as electrolytes in the medium have profound influences on this process. Gelation is a property possessed by lyophflic systems. The gIs are thought to possess a definite structure. the lyophobic colloids are always unstable thermodynamically. other sources of nonideality in a macromolecular solution in addition to the excluded volume. form spontaneously and a re stable thermodynamically. The gels are so firmly set that it becomes very difficult to squeeze o ut any solvent which . Despite inherent instability. while cooling favours gelation. it gradually sets into a more or less rigid mass. gelatin etc. Once the macromolecular chains have become fully extended. exuding a transparent fluid. Avery good example of a colloidal system trans iting into the gel form is blood clotting. (due to solvation of the macromolecule by t he solvent) and increase in the free energy brings the whole swelling process to a standstill. Blood exuding from the wound gets more and rore visc ous to form the clot eventually. leading to a lyophflic dispersion which gets diluted more and more. increasing in volume and setting into a more or less rigid mass. If this process is not checked. This can be exp lained in terms of entropy. On standin g for a sufficient period of time. A balance which results between the decrease in free energy. This decreases the entropy of the system with a concomitant increase in free energy. As the chains extend to their full. This is due to the high solvati on characteristics of the individual long.and clotting of blood axe irreversible.The Colloidal Phenomena 83 is trapped or absorbed within the network of the interlacing fibrils. So me examples of this class are agar agar. Gels may thus be classified into two types. The clot when examined under the microscope appears as a cl ose network of fibrils (fibrin) in which are trapped the blood cells and other liquid compon ents. like silica gel are irreversible An entirely different process may also form gels. swelli ng does have a limit. the process being known as syneresis. Some long increase the limit of swelling . The non-elastic ones. The amount of solvent imbibed may be very large and it is held firmly within the hon eycomb-like structure of the gel. Presence of long in the medium affects swelling. they become more and more ordere d. It continues as long as the free energy of the system decreases. swelling may continue inde finitely. most gels gradually contract. in their s olvent free forms. The elastic gels eve n after complete dehydration can be brought back to their original state by addition of water. elastic and non-elastic. spontaneously imbibe considerable amounts of liquid solvent or solvent va pour. swelling will virtually stop. the ser um. extruding a portion of the dispers ion medium. Gel formation is not always an irreversible process. Lyophilic colloids. solid agar or gelatin formed by the cooling process can be brought back to their original lyophilic colloidal solution forms by gentle warm ing. The clot is a rigid structure but on standing it shrinks. This process is known as s welling. But as observed. Though coagulation of prote in . are made up ofmicelles. It can also be brought about by a change in hydrogen ion activity or by changing electrolyte concentration. In fact. The dispersion finally formed is called the association colloidal system. A micelle is a s pontaneously formed aggregate of micromolecules. the molecules possess both lyophilic and lyophobic groups in the ir structure. though thermodynamically stable. Some gels which are in a semi-solid state can revert back to liquid sol on agita tion. Association Colloids We have a little earlier discussed the nature of lyophobic systems which are inh erently ffnstable and the lyophilic systems which ae thermodynamically stable and are ma de up of macromolecules having great solvation power. There exists another class of collo idal systems which.e. the complete mechanism of muscle c ontraction involves an elaborate gel-sol transition process controlled by the activity of c alcium long. This sol-gel transformation without ch ange of temperature is referred to as thttropy and it has great biological significance. This aggregate achieves colloidal dimensions and is a thermodynamically stable structure. .while some decrease it. The lyophobic components form the core of the micelle (aggregate) while the lyophili c components are on the surface extending their polar groups into the aqueous solution. A striking feature of the association colloids is that the molecules formlng it are amph/path/c in nature i. Viscosity of the gel decreases as swelling continues. On standing the sol reverts back into a gel.. P rotoplasm is said to have thlxotropic properties. 84 Biophysical Chemistry Because of tiffs dual character of its molecules. Examples of this class are the soaps and detergents like sodium dodecyl sulphate. While some are excellent solubflizers o f various types of organic compounds in water. association colloids are of gr eat practical utility. Most of them are surface-active substances. others are good dispersion stabilizers. . In this. if a small amount of the lyophflic system is adde d to the . Protective colloids or p eptlzing as they are often called.. they do not-precipitate out normally.5). Only under abnormal conditions they the form of gallstones and kidney stones. do not form a separate type or class of colloidal syst ems. In other words. t he is protected by the emulsoid. But. The emulsoid therefore. acts in this.They nothing but lyophilic colloidal systems which when added in sufficient quantitie s to a sol hinder its precipitation by electrolytes (see Figure 3. How is a normal person safeguarded pathologies ? The answer lies with protective colloids. the ingredients remainin g the called sensit/zat/on.case as a pro tective An opposite phenomenon occurs if the recipe is altered.85 The Colloidal Phenomena Colloids There are many insoluble constituents in the biological fluids like urine and bi le. o" Water film | a. Aggregates form (1 ml.--" --'.? -'-" ÷ -'"-t "-'.86 Biophysical Chemistry lyophoblc system....--. The suspensold gets sensitized a nd is precipitated out by a very small amount of electrolyte..----*'on of water narge on me t fails . This is because the lyophflic colloid is now enveloped by the lyophobic system facilitating flocculation (see Figure 3.J"-. precipitation is facilitated. Precipitatton t.6). -: ..a' --'.-.. --Na÷ The particle is charged Na÷ due to adsorption of anions . . Albumin or Gelatin Charge on the particle is reduced to zero by the electrolyte added Neutralization of charges lead to aggregation and precipitation .-' -. 10%) Na..-..=. .-......L' . The .5. llustratlon of sensltlzation and protection by a lyophoblc colloidal solution. gum arabic. a nd finally. H e advised a quantitative measurement called the Go/d Number in order to measure the relati ve protective powers of hydrophflic colloids. precipitation. the greater is the protective action of a particular lyophilic substance. The Gold Number is the weight in mgs of protecti ve colloids that when added to 10 ml ofa Zsigmondy gold sol (contains negatively charged hydropho bic colloidal particles).6. albumin etc.ap be used for diagnostic purposes by a clinical chemist. aggregation of the sol particles.gold number of various fluids of our body presents a picture of our health profile and thus c. Examples of protective colloids are gelatin. The smaller the gold number. With the unprotected gold sol. just fails to prevent the colour change from red to blue on the addi tion of I ml of a 10% solution of sodium chloride. Protective colloidal action on a hydrophobic so/. The protective action of various lyohflic substances was studied by Zsigmondy.. & 7 Lyophobic colloidal particle & Lyophllic colloidal particle Sensitization Protection Figure 3.o de thou reduced further proection) is not effective to cause precipitation Protective Gelation Albumin film Fjure 3. the a ddition of sodium chloride brings about charge neutralization. . This p olyelectrolyte will tend to attract the positive long from the dispersion medium around itself. In such a case the origin is said to be internal. The origin of these charges may be either extema/or/nternaL When the charges are acquired b y adsorption of positive or negative long from the medium. Q --Charge due to adsorbed layer of long Q = particleCharge carried by the spherical colloidal . Majority of these biopolymers are elect rically charged. The condenser consists of two concen tric spheres of opposite signs. the macromolecule will soon be surrounded by an/on atmosphere. Electrical Properties of Colloids The colloidal systems we frequently ncounter are the solutions of biological mac romolecules or biopolymers as they are often called. a region in which the re will be a statistical preference for long of the opposite sign. OF COLLOIDS We have earlier discussed the kinetic properties (Brownian movement) and the opt ical properties (Tyndall effect) of colloids. and this is independent of the net charge carried by the macromolecu le. Helmholtz likened this sit uation to the charge distribution in a parallel plate condenser. The charges may be 'strong' or Xveak' depending on the ionization constants of t he acidic or basic groups: The distribution of the charges on the macromolecules may be symme tric or asymmetric. As a result. It may also happen that the charges arise due to active acidic or basic groups present in the inherent struc ture of the macromolecule which remain exposed on the surface. Consider a spherical colloidal particle witha negatively charged surface. and the two plates of the condenser constitute the so called r/g /d doub/e/ayer (see Figure 3.Th Co//o/da/Phenomena 87 " PROPETIF. The behaviour of colloidal systems can be understood properly only when we consider the electrical properties of its constituents as well as the system -as a whole. it is called external.7). r is the radius of the spher e. mobility of the long increa ses and the ion layers become more and more diffused (see Figure 3. Debye and Huckel0 a modified theory was propo sed to explain the charge distribution around a macromolecule. Helmlwltz "Double layer" model. -Q is the charge on the adsorbed ion layer. the long of charge opposite to that of the surface (called gegen/ ons. As the distance from the surface increases. With more contributions from Gouy.theory. from the German gegen meaning against) concentrate and tend to orient as an immobile laye r on the surface.7.8}. and d is the thickness of the double layer. The outer layer of oppositely charged long reduce the po tential on the spherical colloidal particle by -Q/D (r+d). According to this ele ctrical double layer . If +Q is the charge on the spherical macromolecule. th e potential at any point on the surface of the macromolecule will be given by +Q/Dr where D is the dielectric constant of the medium.Outer sphere of oppositely charged long Figure 3. . . Compact layer (surface of shear) ÷ .q+ -#.÷ \.÷ --¢" "----'. The zeta potential has a very close re lationship to the ..-. The electrical double layer in the phase boundaries produces the -potential as a result of lectrostatlc and adsorptive interactions. Qd Dr(r +d) .-v --.. Immobile layer of negatively charged long adsorbed upon the surface from the liquid Fire 3.--" 4.Positively charged colloidal particle I t . The electrical double layer around a spherical colloquial particle whi ch is posvebj charged The net potential between the immobile layer of long and the diffused mobile lay er of long is called the e/ectrok/twttc or zeta potential and is represented by 4.....-.Mobile long in liquid around X. .88 Biophysical Chemistry .../" -. ---\ " -...-..parttc-t-e. = Dr D(r+ = (r +d}-r@ Dr (r+d) " .÷ -r---.8.÷ I---...-" the particle { Diffuse layer) -[L-r.-[ . r" ÷ "" -. the immobile layer. A fl occulate in the form of a clot appears subsequently to stop blood flow. is also called th e Stem layer. During this process. The zeta potential of a sol can be very effectively reduced by addition of elee'rolytes. one of them being blood coagulation. flocculation of the colloid takes place. When this happens. zeta potential is reduced below the critical value (mainly due to the increase in calcium long in the medium) enabling the dispersed particles to form aggregates. i.. . The layer of oppositely charged long whic h is in immediate contact with the colloidal particle. This occurs because the dou ble layer breaks down at the 'critical potential' facilitating coalescence of the particles. The electrolytes decrease the zeta potential to a critical value. The potential between this layer and the particle surface is called the Stern potent /aL The potential difference between the surface of the particle and all the ionic layers in the s olution surrounding the particle is called the electro chemical potential and is represented by E. after which neutralization of the charges takes place resulting in the collapse of the doubl e layer. dielectric constant of the medium and thickness of t he double layer.e. Great slgrtiflcance has been attached to zeta potential in understanding various precipitation reactions of our body. Various other factors which af fect zeta potential are surface charge density. Existence of charged layers on and around a colloidal particle gives rise to man y other potentials besides the zeta potential.stability of a sol. . With the above equation the deduction of the zeta potential by the different types of electrokinetic phenome na (i) By electrOlahore$i: Movement of macromolectles in an electric field may be o bserved measured with a microscopic equipment known as the microelectrophoretic cell. All t he methods the relative motion of the two surfaces in contact.. Th e design [such an apparatus can be seen in Figure 12.3.cm. (9) (Electro chemical . The zeta of any colloidal solution can be calculated by electrokine*ic measurements like electro-osmosis and streaming potential. If oppositely charged electrodes are dipped in a solution of macromolecules.. the double layer is likened to a parallel plate condenser where the plate s are 'd' apart and each carry a charge 'q' per sq. 4ndq C . all the same calculated value of the zeta potential fcr any particular system. The velocity of movement given by . {lO) D D represents the dielectric constant of the medium. various assu mptions have to First..Zeta Potential) The above relationship shows that zeta potential is a little lower than the Stem potential I this is because it is located further out from the surface of the macromolecul e.Colloidal Phenomena 89 The three potentials mentioned above are related to each other by the fo rmula Stem Potential = E . the particles to migrate in the electric field which is thus created. Though the methods are different. For the quantitative treatment of electrokinetic phenomena. The potential difference created be tween the plates may be assumed to be equal to zeta potential which now can be written in form. There will also be a frictional force operating which will tend to drag the solv ent in the direction resulting in retardation of the velocity of movement of the particle.. When the force and the frictional force balance each other the particles move with a less er velocity given by DE 4q .. (13) DE . (11) E = electric field in volts per cm felt by the particle Q = moving charge = viscosity of the medium... (12} or = -- ..law would be : EQ V = -.. e so lvent is seen to se one ofe . e ter flow u e gel tod e negave p ole because e ter acqs a sive chge (it t be mfloned here at e pous gel membe beh aves as enoous set of cap robes). Ife solvent ter. Based on is ely eeent. then V = -r2ED 41I 4Vql { = r2ED . To delve equation reg e -poten to e eleco-osmoc flow. e is pferen adsoon of OH. iO e: In 1808 Reuss t obseed e flow of water mu a membe of powdered qu when a po ten derence was applied.long. en elecc coecon is made beeen e o s of e U-tu ou a p of elodes. In o preous scuion we studied bef e movement of e cooid pcles under e uence of e lecc fld. eoreflc studies elecoecs were made d sever tecques have been ds to study effecve ly e behaour of macromolecules soluon.9). e s of e 'pes' lea e H long to chge e war molecules posively '. measurement of ze pote revolves the measemet of e ve]ocl of e cogod pc]es der pressed e]ecc e]d as e vue of oer components of e equaon (13) be o eas. Dung e flow of ter ou e gel. Original . If e pcles e now forcibly stopd from mog. wch is fact a ckened fo of a lyopc cooid cot mo ve but it c ow e Ivent molecules to svee ou it. : A U-tube is dided to o m compen bya blk of gel ed side e tube (s gure 3. Illustration of electro-osmosls If W" is the volume of liquid flowing through the capillary per second. we see at e dispersion meum now ss mog. appatus c set up as foows." Flow of : : Blek of gel Figure 3.Anode Cathode Solvent 90 Boptujsc Chemtrv us. is movement of fld elecc field is ced ecss or ecoss. e whole to is now a solvent.9.e gel. . 91 length of the capillary. For a parchment membrane or a colloidal gel 'r" and T are very difficult to dete rmine. the volume of liquid 'V" flowing through a membrane is direc tly to the zeta potential and the dielectric constant of the medium and inversely to the viscosity '' and the specific conductivity 'K' of the I/quid. a different term 'K' was evolved. (15} DI The above equation can be used to determine -potetial. E = potential applied in volts.potential and the rate of electro-osmotic flow through a then stands as 4 K rIV = . a . viscosity of the medium. = Zeta potential.The Co//o/da/Phenomena where r = radius of the capillary. 'K' is the specifi c conductance of the liquid in the capillary.. dielectric constant of the medium.= cross-sectional area of the capillary.. E = potential gradient in volts cm-: The relation between the . and is equal to I = strength of current. To clrctunvent. {i//) By streamh potent/a/: A third type of electrokinetic phenomenon observed i s the streaming or flow potential when a liquid like water is forced to flow through A potential develops on either side of the capillary at its two ends (see figure Glass capillary wall . The equation states that at a electr/c current I. these factors. .OH.OH. St reaming is related to zeta potential '' by the equation.OH' H" H* H÷ H÷ H÷ H÷ H÷ H÷ H" H* H* OH" OHOHOH" OH" OHOH" OHOHOH" Glass capillary wall () The nature of the potential depends upon the pressure applied as well as nature of the It has been suggested that the streaming potential results due to forcible flow of liquid tends to separate the oppositely charged layers of the electric double layer.OH. (16} .OH" OH: OH..OH.OH.4 KS PD . = .OH. The hydrophobic colloids (suspensoids) can be flocculated easily by addition of small quantities of electrolytes. and 3 = viscosity of the medium. Normally the potential derived by this method is very negligible. When a colloidal solution is allowed to sedi ment in an ultracentrifuge under the centrifugal force. Another method for study of electrokinetics is the determination of sedimentatio n potential. but it assumes great significance during centrifugation. Precipitation of Colloids The stability of the two general classes of colloids.92 where Biophysical Chemistry S = streaming potential P = pressure applied to the streaming liquid D = dielectric constant of the medium K = specific electrical conductivity of the liquid. The particles move under the impact of a mechanical force. th e gravitational force. a potential difference of measurabl e dimensions develops across the ends of the solution. differs mainly due to two factors. (0 charge on the colloidal particle. it is observed that a point c . the lyophllic and lyophobl c systems. The phenomenon is known as Dom effect after the name of its discoverer. When a dilute electrolyte solution is added dropwise to a colloidal solution and mixed thoroughly after each addition. It measu res the electric potential established by the movement of the colloidal particles with r espect to a Sationary dispersion medium. and (/0 the degree of solvation. pre cipitation does not take place. The reason is that charges on the colloidal particles get reversed s imultaneously so that the particles continue to repel each other and remain separated.omes when after addition of a single drop. This in turn means that the lyophillc systems are more stable . dehydration alone will only convert the emulsoid to a suspensoid but not precipitate it. Both of these wo uld normally bring the particles close to each other continuously. The flocculation values of lyophobic colloidal systems are lower than those of l yophilic colloidal systems. If the electrolyte solution is added rapidly and in excess. The effectiveness of a-salt solution to precipitate a colloidal solution also de pends on the valency of the oppositely charged ion. One is the Brownian motion and the other is the convective movements of the dispersion medium. The hydrophobic colloidal particles are electrically char ged alike and repel each other. The volume of the added electrolyte at this point gives the. Though there are ample cha nces of contact of particles. The stability of lyophilic systems (emuIsoids) lies in the fact that the particl es have a very strong attraction for the solvent in addition to the electrical charge which con fers stability to a sol. it is seen that coalescence does not occur. neutralization of the charges alo ne will not bring about precipitation. with vigorous stirring. These colloidal particles n ow serve as nuclei which grow rapidly by aggregation of other particles finally leading to precipit ation. Higher the valence. rapid aggregation takes place leading to the formatio n of a coarse flocculate.floccula tion value of the colloidal system. This is due to the fac t that the colloidal particles are charged. As long as the particles remain solvated. Similarly. greater is the effect iveness. neutralizing or reversing the charges of the latter. The electrolyte long gets adsorbed upon some of the colloidal particles. Why? Two movements take place continuously in a colloidal dispersion. This property of lyophilic colloids has great biological significance as most . thepower of which depends upon the nature of both the cation and the anion of the salt. Thi s is known as process. Depending on their ability of dehydration of other particles. The reversibility of the precipitate of an emulsoid to the dispersion forri is also vez difficult to achieve and for this reason they are also known a s irreversible .Colloidal Phenomena 93 physiological colloidal dispersions are of the lyophilic type. This seri es determines the i out power of the different electrolytes (see Box 3. Thus addition of large amounts of dehydrates as well as neutralizes the charges on the colloidal particles simulta neously md causes precipitation. Living matter the refore can slight changes in its content of inorganic long without disaster. the long are in series called the /yotrop/c or the Hofmeister series. Effective prec ipitation lyophilic colloid requires the addition of a large quantity of soluble salt.4). 94 Biophysical Chemistry . While OHLong the negative zeta potential H÷ long have an opposite effect.Colloidal Phenomena 95 Besides electrolytes. Thus by varying the v alue of potential to a 'critical potential'. UV radiation. In addition. DONNAN EQUILIBRIUM In a dispersion.is by mutual cancellat ion of charges. The hindranc e may to the colloidal nature of the ion or the electrolyte may be chemically bondedto an like an ion-exchange resin on one side. potentials arise at the junction. Another effective method of bringing about precipitation . precipitation is facilitated. mechanical disturbances like ultrasonic vibrations. This is as mutual precipitation of colloids. showed that when two solutions of electrolytes are separated by a membrane. Compartment B is occupied by a solution of NaCl of 'b'. if the particle is a macromolecule o polyelectrolyte nature. Let us consider a system to be divided into two compartments by a semipermeable water to pass through but not the large long of colloidal In compartment A is present a solution of salt NaR of concentration 'a' where Ris anion of polyelectrolyte nature.also affect the stability of colloids. The for these apparent anomalies was provided by Donnan and therefore the Donnan membrane equilibrium bears his name to this day. Losmotic pressure difference between the two compartments is observed at equilib rium. high speed shaking or stirri ng can the conformation of the colloidal particle and thus precipitate it. Various other physical and heat. The British physicist in 1911. can be done by adding a negative suspensoid to a positive suspensoid or vice-ver sa. NaCl will now diffuse from (B) to (A) and some of it may reversely diffuse . acids and alkalies. H÷ and OH. the genel:al physicochemical propexies may arise. This happens when movement o f of the long through the semipermeable membrane is hindered. back (B). This continues till the system attains equilibrium. rlf 'x' represents the net concentration of NaCI that has diffused from (B) to ( A), then at the situation in the compartments will be depicted as in Figure 3.11 (a and b). (A) (B) Na÷ (a mols) Na÷ (b tools) () CI(b mols) {a mols) Figure 3. I i. (a) Distribution of ions.across a semipermeable membrane at zero time. (A) (B} Na÷ (a + x mols} (a mols) Cl{x tools} Na÷ (b x mols} CI(b - x mols} Figure 3.1 I. (b) Distribution of long across a semipermeable membrane at equili brium. 96 Biophysical Chemlstr The rate of diffusion of NaCI from (B) to(A) is proportional to the product of t he concentration of Na÷ and C1- in (B) and the rate of diffusion of NaCI from (A) to (B) is proport ional to the product of the concentrations of Na÷ and CI- in (A). It might be mentioned here th at in very dilute solutions, activity terms can be replaced by concentrations. The situatio n is represented as below : [Na÷]B [CI-]s = [Na÷]A [CI-]^ to-x) to-x} =(a+x} (x} The above equation shows that while in compartment B the concentration of Na÷ and CI- is the same, in compartment A the concentration of CI- is less than the conc entration of But as electroneutrallty requires the presence of equal numbers of cations and a nions in each compartment, it follows that [Na÷ls = [CI-] and iNa÷l^ = [R'l^ + |el-|^ where [R-I^ is the concentration of the restricted ion in compartment A. The abo ve equation points out the fact that the concentration of the diffusible anion is less in th e compartment containing the non-diffusible ion than in the other compartment. The restriction of the nondiffusible anion at the membrane while the cation is tending to diffuse through it, sets up.a potential difference between the two solutions. At equilibrium, the solution in the compartment A acquires a positive potential with respect to the other because of the slight excess of Na÷ long. Thus we have seen that the presence of a non-diffusible ion on one side of a dia lyzing membrane causes unequal distribution of every diffuslble ion on the two. sides of the mem brane. While the positive diffusible long concentrate in the compartment containing the negative non-diffusible ion, the negative diffusible long are more to be seen on the opposite side. The relative concentrations of the non-diffusible and the diffusible long is important in a s ystem in Donnan equilibrium. If the concentration of the non-diffusible ion is increased, a grea ter inequality in ion distribution is observed. This leads to a further increase in membrane poten tial. On the other hand, the greater the concentration of diffusible long, smaller is the mem brane potential due to the masking of the effect of non-diffusible long. Therefore, the Donnan e ffect can be decreased considerably at high electrolyte concentrations. The osmotic pressure difference observed at equilibrium due to inequalities of d istribution of diffusible long in the two compartments can be minimized by dissolving the po lyelectrolyte in a salt solution of high molarity (0.1 M or higher) and then using the same salt solution in the other compartment. Another modification which can be adopted for protein solutio ns is to select a pH very near to the isoelectrlc pH of the protein so that the net prote in charge is reduced to a minimum value. Under these conditions, the flow of the oppositely c harged diffusible ion across the membrane and its concentration in the compartment containing the protein molecules will be decreased. The osmotic pressure in a system exhibith-lg Donnan effect can be calculated by the formula P--cRT where c = concentration of the long R ffi gas constant T = temperature At 27C, the osmotic pressure of a system of the following type can be calculated thus : 97 5.10 mols The Colloidal Phenomena Na÷ (a) R{B} Na÷ {b} Cl- {b} (at zero time) Semipermeable membrane Let a = 3 mols and b = 4 mols, then amount of NaCI diffusing from (B) to (A), re presented by 'x' will be, X a +2b This is so because at equilibrium , [Na+]s [CI-]s = [Na+]A [CI-]A or or or or (b-x}(b-x} = {a+x}(x} b2-2bx + x2 = ax + x= b== ax + 2bx b= = x {a + 2b} b= X a+ 2b Putting the value of a and b n the above equation, we get (4)2 16 16 = 1.45approx. x - (3 + 2 x 4) - 3+8 11 .. Now, calculating the concentration of each of the long in the two compartments w e Na÷ (a.+x) = 4.45 {B) Na÷ (b-x) = 2.55 CI(x) = 1.45 R(a} = 3.0 8.90 tools Cl(b-x) = 2.55 Since the osmotic pressure is given by the equation, P =.cRT 98 Biophysical Chemistry by substituting all the values we get, P = (8.9 - 5. I) x 0.082 x 300 (as 27°C = 300K) = 3.8 x 0.082 x 300 = 93.48 atmospheres Biological Significance of Donnan Membrane Phenomenon The Donnan effect plays a significant role in our body because of two basic stru ctural organizaUons. (0 Most of our body membranes are dialyzing membranes, i.e., they allow the movement of water, gases like O and COa, long and small organic molecules across them whi le disallowing colloidal particles, and (//) The non-diffusible protein anions are present in large quantities in cel ls and in plasma but not in interstiUal fluid. Both the above factors promote unequal distribution of diffusible long on either side of biological membranes which in turn would result in establishment of a pH differe nce and.an osmotic difference between the compartments. Minor pH changes are tolerable for most of the tissues but drastic pH changes may pose problems. The body saves itself from the h .armful influence of the latter with the help of its buffer systems. But sometimes maint enance of a drastic pH difference is a necessity, as is in the case of stomach. Gastric dige stion cannot proceed ff the pH is not maintained at a very low level. In this case, the gastr ic mucosal membrane gears up to permit the hydrolysis reaction PCI + HOH POH + HCI where P represents the non-diffusible protein long. The H÷ long, with associated C I- long (to maintain electroneutrality) will move from gastric mucosa to the gastric lumen b ecause of the non-diffusible P* long in the protoplasm. This leads to a greater concentration of H÷ long in he lumen and subsequently the pH of the gastric secretion fails to very low values. The Donnan effect applies to all kinds of diffusible long. The red blood ceils p rovide another case where Donnan effect determines the distribution of long between the content s of the cell and the surrounding plasma. The erythrocyte membrane is freely permeable to HCO and Cl-anions but is impermeable to the protein haemoglobin which exists in the anionic form. When blood flows through capillaries, HCO diffuses into the plasma by passive diffusi on. About 70% of the HCO formed in the red cells enters the plasma. Na md K÷ are n ot available for free diffusion due to the operation of sodium-potassium pump. T o maintain electroneutrality, CIfrom the plasma enters the erythrocytes. But at equilibrium the Cl- ion concentr ation in RBC and plasma is not found to be equal. Only 70% of the CI- long present in the blo od plasma enter the cells. The unequal distribution of this diffusible ion is mainly due to the Donnan effect. As the RBCs of venous blood has more of riCO long, there is a greater concentration of Cl- long in these RBCs during this exchange phenomenon. The above examples have made us understand the usefulness of Donnan membrane equilibrium in biology. However, there are cases where the same effect becomes a bane to the system. An example that can be cited is that of oedema in ttssues. When the plas ma .albumin content falls below the normal value, salt and water retention tares place in th e tissues. The movement of the concerned electrolytes in and out of the ceil membranes occurs m ainly due to the Donnan effect. Other developments like increased secretion of a ldosterone and vasopressin fotlow soon to result in a full-fledged oedema state. ne CoY.ol Phenomeng 99 The Donnan equilibrium has played a very important role in interpretation of oth er physiological phenomena like absorption of glucose by the intestine, the distrib ution of diffusible long between blood and the cerebrospinal fluid and in urine formation in the kid ney. Besides its role in physiology, it is also being exploited for the benefit of ma nkind. Kidneys, apart from being one of the main organs involved in acid-base regulation, also e xcrete many toxic substances through urine formation. Naturally, both these functions are al tered in kidney pathology with life-threatening repercussions. For such patients, artificial kid neys are used. These artificial kidneys have dialyz'ng membranes which form minute channels for blood on one side and a dialyzing fluid on the other side. The thin dialyzing cellulose m embrane allows all constituents of blood to .diffuse freely in both directions with the excepti ons of plasma proteins and cellular components. In this way, the toxic substances accumulated in the bl ood can diffuse through the cellulose membrane into the dialyzing fluid. This diffusion can be tailored as per the needs of the patients by altering the concentrations and compositions of the non diffusible long. It is obvious that Donnan effect plays a major role in the,functioning of artificial kidneys. Suggestions for Further Read/ng I. Edsa]l, J.T., The S/ze, Shape and Hydration of Protein Molecules in The Proteins, Vol. I, Part B (H. Neurath and K. Bailey, eds.), Academic, New York, 1953. 2. Haschemeyer, R.H., and Haschemeyer, A.E.V., Proter: A Guide to Study by Physfcal and ChernfcaIMethods, John Wiley and Sons, Inc. 1973. 3. Ward, A.G., Co[[ofds, Their Propertfes andApplfcatfons, Blackle and Sons Ltd., London, 1948. 4. Me Ba/n, J:W., Co//ok/Science, Reinhold Pub]/shlng, CoG., New York, 1950 . 5. Kruty, H.R., Co//ofd Sdence, Elsevier Publishing Co., New York, 1952. 6. Adamson, A.W., Physical Chemistry of Surfaces, Intersc/ence Pub]/shers I nc., New York, 1960. 4 DIFFUSION AND OSMOSIS Matter tends to move spontaneously from a region of high concentration to a regi on of lower concentration until the composition is homogeneous throughout. This moveme nt of molecules without any energy expended is called passive transport or diffusion. The molecules in a solution are in a state of continuous motion as a result of b ombardment by solvent and other solute molecules if present. The direction of movement is c ontinually changing in a zig-zag fashion. The velocity of this movement, also called Browni an movement (see Chapter 3) is given by the equation, M ... (1) where C is the root mean square velocity in cm sec-, 'R' is the gas constant having a value of 8.314 × 10 ergs/degree/mole, T is the absolute temperature in Kelvins, and M is the molecular weight of the moving particle. Due to Brownian movement, the displacement of the molecules per unit time is not in a straight line. The small molecules and long (crystalloids) diffuse rapidly while the macromolecules owing to their large size and shape have a sluggish motion. Another factor affec ting the movement of molecules is the concentration difference. Greater the concentration gradient , more rapid will be the diffusion. Biomembranes are selectively permeable in nature. Diffusion of solutes across th ese membranes is mainly of two kinds : (/) Simple diffusion, (//) Facilitated diffusion. Simple diffusion may take place directly through a lipid bilayer, through "holes " created in the membrane by membrane proteins or by discontinuities in the lipid bilayer. Bu t there is no active participation of any membrane protein in this type of solute movement. On the other hand, facilitated diffusion compulsorily occurs with the participat ion of a carrier molecule, usually a membrane protein, It may perform its task in any of the three ways mentioned below : ((} it may move across the membrane with the substance to release it on the other side and then return to its original position to continue the cycle, or Diffusion and Osmosis I01 (//) a conformational change may take place in the transport protein on bindi ng to the solute which may help in the translocation of the latter to the opposite side, o r (///) the solute may be shuttled from one membrane protein to another till it reaches its destination. A MOLECULAR - KIITIC APPROACH TO DIFFUSION Diffusion is a process of equilibration thatis directed along a concentration gr adient from a region of higher concentration to a region of lower concentration. At equilibr ium the molecules will be uniformly randomized throughout the system leading to an increase in ent ropy. It is therefore a spontaneous process, according to the second law of thermodynamics. Due to the random walk of individual molecules owing to vibrational and thermal motion, it is difficult to measure the actual displacement per unit time for each molecu le. However, a statistical consideration of all the diffusing molecules as a whole shows that a t each point time a net flow exists, which is proportional to and follows the concentration g radient. This rate of net movement of molecules can be described by Fick's laws of diffusion. Fick's Laws of Diffusion Let us first assume that the solute molecules are moving in one dimension only. A molecule situated at the origin initially is capable of moving either to the left or to t he right in distances of average length W. We shall call these small displacements as 'steps'. The net movement of a molecule can now be given by the excess steps taken to the right over the left o r vice versa. The probability of the steps taken in either direction remains the same. If 'n' is t he total number of steps covered by the molecule in unit time, the total distance travelled will be equal to 'r', 'n' is constituted of the steps taken by the molecule to the right and to the left indi cated by symbols 'mr', and 'm,' respectively. As already stated that the chances of a leftward or rightward step are equal, the most probable end point of any particular molecule will be at the ori gin itself, i.e., rn is equal to mr, therefore rn-mr is equal to zero. However, the system contains a large number of molecules and some of these molecules might move excessively to the left or to t he right. As the time increases, the total number of steps 'n' also increases and with it increas es the possibility of more and more molecules moving away from origin by purely random motions lead ing to diffusion. Rather than one, let us now consider a model having two origins separated by a p lane. From both the origins molecules radiate out into the solution across the plane l ying between the two origins. Naturally such a variation would change the concentration profi les of not only the two origins but of the plane also. From the above paragraph we know that the change in the concentration profile depends upon the total number of steps taken by the molecu les (n), distance covered (.) and on the initial concentration. Therefore, the net displacement pe r unit time across the plane in any one direction will be determined by the difference in th e initial oncentration of the two origins and by 'n' and W. This is the molecular basis of lck'sfirst law of diffusion which is in effect described by rate equation. It states that the a mount of solute 'da' diffusing across the areaA (1 sq. cm) in a length of time 'dt' is proportional t o the concentration gradient dc/dx at that point, or ' ... where D is a constant of proportlonallty, also called d{1sion lent. It is indepe ndent of the concentration at 'x' and x + dx, and related to 'n' and ',' mentioned above. The negative sign arises since diffusion takes place in the direction of decreasing concentration gradient. ... (3) where 102 Biophysical Chemistry While formulating the first law, Fick assumed that the laws of diffusion were an alogous to the news governing the transfer of heat derived by Fourier earlier. METHODS OF DETERMINATION OF DIFFUSION COEFFICIENT (D) One of the methods to determine 'D' is to measure da/dt, also called the flux, a nd then find dc/dx frbm it. From this value we can obtain D as the flux obeys Fick's fir st law. Due to diffusion the composition and therefore the refractive index of thesolution chan ges. Most of the methods for measuring rate of diffusion involve measurement of such changes in t he refractive indices with the help of such techniques as light absorption, fluorescence and T yndall effect, More recenfly the interferometric methods of studying the boundary changes have been preferred. Experimentally, the measurement of diffusion constant is done by two methods : (/) the porous disc method and (//) the free diffusion method. (0 In the porous disc method the particles are allowed to pass through a porous disc made of sintered glass or alundum. A concentration gradient is thus created across th e porous disc. A solute is now chosen whose diffusion constant is known and its rate of diffusi on is measured by this method. The molecule whose diffusion constant has to be evaluated is now treated in identical manner and its rate of diffusion through the porous disc is measured. From the obtained data, the diffusion constant of the unknown solute molecule is calculat ed. The value of 'D' thus obtained is of a relative nature. For absolute measurements of'D' by the free diffusion method, Fick's second law of diffusion has to be applied. It is a partial differential equation derived from Fick's fir st law of diffusion. The simplest assumption during appication of this second law is that the diffusi ng particles do not participate in any chemical reaction within the diffusing space. This law ca n be expressed by the equation, de where - = rate of change of concentration with time d2c and- = first derivative of concentration gradient with respect to distance. For three dimensions, the equation becomes / x, y and z are the space variables. The above equation has ALSO been expressed a s de B2 --- DVc ... (5) dt Diffusion and Osmosis 103 -2 is the Laplace operator, in this case operating on 'c'. To use equation (5), it has to be where We solved first. The solution takes into consideration the boundary conditions and initial conditions operative in the system under investigation. These conditions determine which of the multitude of possible solutions of a differential equation is applicable to the system. To simplify the mathematical dealing, one may employ a specially designed diffusion apparatus wh ich is geometrically simple or use an analytical centrifuge The spread of an initially sharp boundry between the solute and solvent is observed as the solute diffuses into the solve nt layer. Diffusion in biological system is, however, mostly associated with chemical reac tions. In such situations the. simplest assumption that can be made regarding the diffusin g system is that within the diffusing space the rate of utfllzation of the diffusing solute is constant, i.e. independent of time and place. Equation [5) in such conditions will take the for m -- = DVc - A ... (6) dt where 'A'. represents the rate of consumption. Another frequent observation in b iological system is that due to stationary boundry conditions the concentration is independent of tirne at all positions of the diffusing.space. Under this condition, dc/dt = 0 and equation ( 6) becomes, D.Vc = A, assuming again the constant consumption of the diffusing solute. SIGNIFICANCE OF DIFFUSION COEFFICIENT The speed of movement of a particle is characterized by its diffusion coefficien t which is a function of the size and shape of the molecule. Knowledge of the diffusion coeff icient is necessary for ultracentrifuge studies as it provides us with information regarding the sha pe of the particles in solution. Perhaps it is of more importance during the determination of moecul ar weight of macromolecules as there is a distinct relationship between the molecular weight of a substance and its diffusion coefficient. Before we proceed to the mathematical treatment f or obtaining the relationship between molecular weight and 'D', we shall discuss the molecular na ture of a liquid film. The /atttce theory explains beautifully the molecular nature of a liquid film. According to this theory the liquid film appears as a network of molecules with empty spaces in between (see Figure 4. I). These empty spaces or 'holes' as they are called are the regions where molecules are trapped during their translational movement. Molecules thus move from hole to hole, creating in the process new holes to trap other molecules. This movment of molecules from one hole to another is called diffusion. However, since the molecules of a liquid are attracted by other molecules adjacent to them, they are unable to move swiftly in the holes created for them. Therefore, in order to move a molecule into a hole energy has to be expended. IfaE is the total energy required to break the bonds a molecule makes with its nelghbours to flow into an adjacent hole, will represent the dist ance travelled by a molecule to fall into a hole. If the initial concentration o f the sample at the origin is 'c', on 104 Figure 4.2 'dc. . +dx Distance travelled (.x) Diffusion of molecules across an energy barrier. , is the dstance between successive equtltbrrn posons, and aE Is the total energy required to enable a molecule to free from its neghbours and j'Iow into an ¢acent hole Bophsca Chemstr diffusion of the molecules through a distance dx the concentration at the new equilibrium position dc would change to c + - (see Figure 4.2). Therefore, in order to move into an adjacent hole the molecules should possess an energy equivalent to or greater than AE. Not all molecules possess AE. The fraction which does possess AE can be represented by the number e-/R, according to the Maxwell-Boltzmann distribution law. As time 'dr' lapses, some of these molecules will acquire the most favourable orientation to move faster into the hole, so that the total number of molecules per unit volume diffusing from left to right will be depicted as N = cQe-/T where Q = the number of molecules possessing the correct orientation. Similarly, the ones crossing over from right to left will be given by The net movement from left to right direction would now be Nm = N- Nr or Nm = -X --.Q.e-E/T per unit volume ... (7) dx If the diffusion has occurred across a plane of area 'A', the net total number o f particles that have corssed over from leil to right along the concentration gradient per u nit time would be given by Nm ..2A.Q.eA/RTdc -... (8) dt dx Comparing eqn. (8) with eqn. (3), we see that D = ,2.Q.e-Z/RT " Thus, if the values X, Q and E are known, D can be easily obtained. 4n r3N 3M ... (13) Di.Jiusion and Osmosis 105 The hydrodynamic properties of a molecule are strongly related to its size and s hape. Diffusion is of two main types -- translational and rotational. Out of these, th e latter is characteristic of the type of molecule. A large spherical molecule will have a s ingle rotary diffusion coefficient. An ellipsoid of revolution is characterised by two coefficients as it possesses two axes while a general ellipsoid molecule will have three coefficients, one for ea ch of the axes. These rotary diffusion coefficients of macromolecules of various shapes can be d etermined by flow birefringence (see Chapter 9) and by electrical methods if the molecules ar e dipolar in nature. Einstein in 1905 gave a relationship between diffusion coefficient and radius of a macromolecule on the basis of Stokes' law of diffusion. The assumptions for this relationship are that the size of the diffusing molecule is many times greater than that of t he solvent molecule and secondly, the solvent is a continuous medium. Einstein provided the relationship between diffusion coefficient (D) and frictio nal coefficient (f} as D =-... (10) f where k = 1.38 x I0-=3 JK- (Boltzmann's constant) and T = Temperature in Kelvin. The coefficient T depends on the shape and size of the macromolecule, but not on its mass. For spherical particles, Stokes provided the relationship f=6lr ... (11) where r = radius of the particle, and -- viscosity of the solvent. Combining eqns. (10) and (11) we get the Einstein-Stokes' equation as D = (12) 6xrlr ... 4 Since the volume of a sphere of radius 'r' is -- ra, and the volume of a spheric al molecule is M; 3 -- where ; is the partial specific volume, M being the molecular weight and N th e Avagadro's N number, we can write 106 ... .., (16) RT Do = -- (as 'K' .the Boltzmann's constant is Nfo equivalent to the gas constant per molecule, it Is replaced by R/N) where fo is the frictional coefficient of a spherical particle. Or, Do = As the macromolecules being studied are of various shapes, the experimentally de termined values of'D' are usually smaller than those calculated theoretically on the assu mption that the molecules arespherical in shape. The ratio f/fo i.e., the ratio of the frictiona l coefficient for nonspherical particles (f) to the frictional coefficient of spherical particles (fo ) is termed asfr/ct/ona/ rat/o or dtssymetry constant. The value off/fo for spherical particles is 'one' and as the deviation from spherical shape increases for macromolecules, the value of f/fo becomes pro gressively greater. DIFFUSION OF ELECTROLY'rEs Earlier we have discussed the zlg-zag movement of molecules in solution on accou nt of Brownian motion. However, the same theory does not apply to solutions of electro lytes. The long here are influenced by the electric field and the net movement is according to the sign of their charge. One ion attracts around itself other long of opposite sign and ten ds to drag them along with itself. As a result, long of opposite charge do not move singly but r ather in the form of pairs. A faster ion is thus slowed down by its 'sluggish' partner while the l atter is speeded up by its association with the former. Thus it is difficult to obtain the diffusion coefficient of any single ion. What we find actually is the diffusion coefficient of the ion pair, If an electric connection is made after the solution of electrolytes has come to diffusion equilibrium, a flow of current will be observed. This is due to the dissociation of the ion pairs which under the in fluence of the extemally applied electric field now move independently to their respective oppo sitely charged electrodes. It is now possible to measure the ionic mobility of individual Long. Each ion will have a different ionic mobility and the mobility is a measure of t he speed of the long as it moves through the solution. Therefore, there is a distinct relati onship between the mobility of an ion and its diffusion coefficient. It is given by ) RT (v, + v2) D° = " ... (171 N Vla2 + v2D where D° is the diffusion coefficient of the electrolyte at infinite dilution, N i s the Avagadro's number, R is the gas constant equivalent to 8.314 x 107 ergs/degree/mole, T is t he temperature in Kelvins, bt and 12 are the absolute mobility of the two oppositely charged lo ng and v and v2 represent the number of positively, and negatively charged long respectively obt ained on dissociation of the electrolyte (for e.g., ff one molecule of NaC1 dissociates i t will give rise to one positively charged ion, Na÷ and one negatively charged ion, CI'. Similarly CaCI2 w ill give one positive ion Ca2÷ and two negative long of CI'}. The value of 'D' obtained by equation (17) is of limiting nature as it is obtain ed on the assumption that the electrolytic solution is infinitely diluted. However, at low concentrations, But ff it In a lipid layer. (18) where 7± represents the "mean ionic actlty" coefficient and 'c' the concentration of the solution. the diffusion coefficient will be reduced in lipid solvent solutes. all the hydrogen bonds have to be broken together which will r equire a mergy. The equation now takes the form : . Therefore.'discuss subsequently.e. The lipid layer acts as an effective diffusion barrie r. Biological membranes have a lipid bflayer sandwiched between two layers0f proteins. Now if the molecule has to move in water . If all these bonds have to be broken when the solute moves.the model provided by Davson and Dani elli. At higher concentrations the above equation has to be modified further to take a ccount of the solvation of long which we shall. its hydrogen bonds haveto be broken. move from hole to In the lattice... The energy required to brea k one bond is approximately 4. A stable solution is one in which the solute-solvent bonds are of the same order of magnitude In the same manner an aqueous solution will be stable only ff the is capable of forming hydrogen bonds with water in a manner similar to the hydro gen between the water molecules.2 kcal at 27"C. give an explanation on diffusion of :solutes across bio-membranes. D=DO +(l+dln¥+ / ) . We sh all discuss them one. Some solutes form many such hydrogen bonds with water molecules.Dtffuslon and Osmosis 107 the actual value of D can be found by taking into consideration the activity of the solution. Having gained knowledge about the various types of interactions in a solution wh ich affect and on the basis of lattice theory we can now. i. When the solute has to diffuse.. It will be appropriate to mention here some struc tural characteristics of living membranes as per. . the lat ter providing integrity to the membrane. Several reasons have been attributed to this behaviour of the lipid layer. then diffusion will be slow . all hydrogen bonds that it makes with water need not be broken at the same time. Cations are slow in diffusion than the anions because of their bulk. Diffusion thus is retarded. the diffusion rat e of a is lowered when passing through a lipid medium. (10 The lipid bilayer is a closely knit structure with intermolecular hydrophobi c bonds acent side chains of the fatty acids making it even more difficult for a molecul e to it. the . a large amount of energy to be expended in breaking the hydrophobic bonds.(0 A molecule has to break all its hydrogen bonds with water if it has to p enetrate the lipid In an aqueous environment this would not have been difficult as the H-bonds coul d be one at a time and similarly reformed one at a time. For a lipid membrane 50A thick. diffusion is slowed down because larger holes h ave to be by displacement of a large nnmber of solvent molecules. Therefore. lesser is the diffusion. (li0 For large polyelectrolytes. To create even a single hole for a moving solute molecule. les ser is value of 'D' as can be seen in equation (12). Larger the molecule. (iv) A hydrated molecule has to shed its bound water molecules to penetrate into the lipid again requires a considerable energy. the resistance becomes too much and diffusion is reduced extent. Greater the de gree of a molecule. The values of spherical molecules are gr eatly affected by hydration. . It ca n happen when two solutions of the same electrolyte at different concentrations are separated by a membrane and also when two different electrolytes of the same concentration are kept sepa rate due to the membrane. and are a characterist ic of the membrane as well as the long. (19) Ceb 108 Biophysical Chemistry bulk being chiefly contributed by the water molecules around them.of a single concentration separated by a membrane.. Two equations have been provided to calculate the diffusion potentials of membra nes. ^. there are several sophisticated membrane processes affecting diffusi on of solute molecules in and out of the cells.and C÷B.058 laA+ ]an logt . As a result a potential is set up across the membranes through which these long diffuse. They are : Case a E = 0.5. Hydration cau ses a swelling of the molecule by binding water. and thisincreases the frictional effect. If the membrane has the pores of a size that would not allow entry of one particular ion through it.ti¢. (v) Lastly. These potentials are called diffusion poten'. . the diffusion potential will be high. The anions and cations of an electrolyte solution move at different rates. For m acromolecules ff the frictional ratio is more than 2. (20) 1C + laB C2 when there are two different concentrations C and C= of the same salt across a m embrane.058 log laA + lab c + ga when there are two different salts A÷B. the effect of hydration becomes negligibl e.. and E = 0... B. However. The above equation tells us that the relative mobilities of the diffusing long a nd their concentrations are the factors determining the magnitude of the diffusion potent ial developed across a membrane. B and C respectively. these potentials are unstable as concentration diffe rences are bound to occur on further diffusion. . c are the ionic mobilities of the long A. Some of these movements require energy and thus are termed active others just move on across the gradient and therefore called passive. Osmosis takes care of both of these. The value of K for oxygenin human beings. We can demonstrate A glass tube open at both ends is chosen and at one end is tied an membrane in the form of a sack. For it is semipermeable to small long and molecules but not big colloidal particles.capacity for oxygen. Osmosis is one of the major exchange processes which is to the living body and therefore has to be regulated carefully. across a semipermeable membrane till the equilibrium. excluding others.This is due to the physiologicaldea d space. lungs. The rate of diffu. This flux of metabolites is associa ted with of solvent. the one to go out also has to face scrutiny.. It is not only t he of the various long in the body fluids but also their content which is important for functioning individual. though. by the DIFFUSION OF WATER ACROSS MEMBRANE : OSMOSIS So far we have discussed the free diffusion of molecules and diffusion of solute s across process which is complimentary to the ones described above and obeying mechanism is osmosis. Osmosis be longs to It is concerned with the spontaneous flow of solvent only. The semipermeable nature varies from membrane to membrane. while some may be totally Biological membranes. By definition it s theflow a dilute to a concentrated solution. llng area and the istance through which diffusion occurs.109 Due to and Osmosis always foundto be less than the expect value. that which is needed. are mostly specific. The anatomical dead cpacity. They have a vast range of systems embedded within them which will carefully 'sniff the metabolite to be while it waits outside and only allow one kind to enter. Now the tube is half-filled with concentrated su gar . for others be differentially permeable to certain long.can be determined from measure ments of diffusio. .3).in a beaker of water (see Figure 4. After some time we observe that in the tube has risen. The level of the liquid rises until the hydrostatic press ure so sufficient to stop the flow of solvent. or osmosis. into the tube. This hydrosta tlc developed as a result of osmosis is called the osmotic pressure of the solution and is as the excess pressure that must be applied to the solution to prevent the passa ge of the t into it thorugh the semipermeable membrane separating the two solutions. The hydrostatic pressure so developed in the containing the solution is enough. osmotic pressure is one of the four. When a solute dissolved in a solvent. the search began for an ideal semipermeable membrane.Animal membrane ---4 (semipermeable) Figure 4. the energy of the solvent molecules is reduced considera bly the solute-solvent interactions. Traube in 1867 membrane of a gelationous precipitate of copper ferrocyanide. Cu2Fe(CN)6 for low weight solutes in water. semipermeable membrane rapid movement of the solvent molecules across the barrie r in the direction of the solution. Why Exactly Does Osmosis Take Place ? The answer becomes very simple if we look at it thermodynamically. M. on. For high molecular weight solutes in organic solvents. colligative properties that a solution poss . made of cellulose.3 Osmosis through animal membrane The osmotic behaviour of molecules was first reported by Abbe NoIlet in 1748. greater is the osmotic pressure deve loped.= -. When the free energy of the solvent molecules in the solution is resto red to equal to that of the pure solvent.---. More number of solute particles in the solution. cellulose nitrate or animal membrane have been found suitable .Water i" -.Initial level -----* Sucrose solution I---.--. an equilibrium is achieved and osmosis stops.110 Hydros pressu Btophysicat Chemistr Final level h. When this solution is separated from the pure s olvent by'. to increase the free energy of the solvent mo lecules in solution. Osmotic pressure of the filtrate devoid of protein constituents and the raw (unfiltered) serum sh owed that proteins gave osmotic pressures of 30-40 mm Hg. directly proportional to the absolute t emperature and concentration. With a membrane impermeable to protein but permeable to osmotic pressure.esses pressure. namely. Under conditions it is just like gas pressure. Since then. and is independent of the chemical nature of the dissolved materi al. are prepared in dilute sa lt If the membrane is impermeable to both salt and colloidal particles. in general. rise in boiling point. . When working with a colloidal solution. depression of freezing point. the renal function the secretory processes. we obtain t ota/ pressure. various attempts were made to design the os motic measurement apparatus to a type where the osmotic pressures of the biological not be affected adversely by extraneous factors. being the others) . This experiment provided an insi ght osmotic behaviour of cells in various physiological processes. Protein solutions. mention has to be made about the nature semipermeable membrane. The latter is useful while deducing the molar mass of the coll oidal particles MEASUREMENT OF OSMOTIC PRESSURE The first determination of osmotic pressure of macromolecular solutions began wi th in 1899 He filtered blood serum through a semipermeable membrane made of successfully freed the serum from its crystalloid constituents. paraffin. The osmotic event was r apid :the rise due to capillarity in themanometer tube required several days to reach an equilibrium osmometers designed later were easy to handle and more accurate. Later. toluene or alcohols with could be attained in a very short time.and Osmosis 111 Sorensen in 1917 first published the data of his measurements of osmotic pressur e of solutions under carefully controlled conditions.Rowe and Abrams in 1957. could be operated without thermostatic control and the sample for determination was small volume (only 0.5 rnl). The sample requirement also was low. this problem s circumvented by the invention of an electronic osmometer by Rowe andAbrams in 1957. The U. used by Sorensen and others in the first half of 20th though efficient. instruments required a rigorous thermostatic control within 0. The manometers these latter type of instruments contained organic liquids.osmotic pressure using ample was designed as above by.004o. His results and those of Starli ng's on which was reproduced using collodion membranes and better designed modem showed that a strict control of pH and salt concentration. A detalled discussion of the apparatus is given in pressure (P) Transducer Electrical cireuit Galvanometer An osmometer adaPted for rap dmeasurements of. took several days for standardization. and knowledge of the of protein are mandatory while determining the osmotic pressures of protein The osmometers which were.tube has platinum foil which isconnected . due to small pore size membranes. will flow from A to B because the osmotic pressure of the solution in B is great er than in There will also be a diffusion of sugar molecules from B to A at a very slow rat e. ASOLVENT The significance of o is better understood when we study-the osmotic behaviour o . symbolized by o. However. Let us consider a vessel fillet and divided into two compartments (A and B) by a porous membrane. though the movement of the solute molecules will be impeded.o = 1 A SOLUTE Biophysical Chemistry: OSMOTIC BEHAVIOUR OF CELLS We rarely come across biological membranes which are perfectly selectlvely the solvent and exclude solute molecules completely. the effective fraction of the area on the membrane availa ble for diffusion.. If a solute like sugar is now added to compartm ent B. i. ASOLUTE approaches zero and o approaches I. the second half of the equation. in the osmotic pressure equ ation. The membrane impermeable to solute molecules. there a very slow diffusion of low molecular weight solutes. If AsoLv represents the effective fraction of the area on the membrane available for diff usion particles and ASOLVET. so that neff = The reflection coefficient has an inverse relationship with permeability coeffic ient. It depen&¢ on the nature of the selectivly permeable membrane and the nature of the solute. then If the pores in the membrane are of size which would not allow the solute molecu les to diffuse. Due to this. The effective osmotic pressure of a solution can be calculated by including a te rm Staverman's reflection coefficient. effective osmotic pressure as judged by the movement of solvent from A to B will be the osmotic pressure calculated from the solute concentration alone.e. Under these conditions. This leads to the inflow of some solute molecules along with the solvent while others are reta ined.f living cells. there is no unidirectional flow of solvent and the cell size remains the . If the cells are placed in a medium whose osmotic pressu re matches that of the cell contents inside. Living cells have selectively permeable membranes which are very permeabl e to water and some low molecular weight solutes. This fraction of the total osmotic pressure o f a solution is termed as its ton/city. the solution exhibits only that fraction of its total osmotic pressure that is due to the solutes which are retained. but much less permeable to other substances. the milliosmol (abbrevia ted .e. it is convenient to use a smaller unit. If the same erythrocytes are suspended in 280 m m . greater osmotic than another. In osmometry. grams per Iitre though are preferable because of the inconvenience of weighing liquids . one mol of glucose.. a solution is said to be hyperosmotic and hyposmot/c if it exerts a lower The osmolarity of a solution is best expressed as osmoles. O n the other hand. each ion contribut es the same molecule. Water then will be a bstracted from the cell resulting a shrinkage of the cell. the total being 3 long. one mol of sodium chloride (NaCI) will produce 2 osmo les long while one mol of calcium chloride (CaCI) will PrOduce 3 osmoles s it dissociates into one cation and two anions. The reflection for mannitol is approximately equal to 1. and therefore mannitol at 280 mM is isotonic for red blood cells. a cell will swell if it is placed in a dilute medium because the osmotic pressure of the cell Contents being higher will cause the cell to absorb water from outside and increasein siz e. which refers to increase or decrease in size of ceils. its osmotic pressure is greater than that of the cell interior. The medium then is said to be hypotonic. The medium then is said to be isotonic with the cell. and this is unaffected by temperature. concentrat ions should always be expressed as molal concentratlons Molal concentrations are expressed i n terms of weight of the solvent. The osmolarity of the erythrocytes is approximately 280 mflliosmoles. Ideal solutions o f equal molarity exert same osmotic pressure and therefore they are called/sosmotic. If the medium is con centrated. osrno/adty refers to the concentration of solutes in the solution. which equals 1/1000 of an osmoL The milliosmol is equivalent to a solution of I millimole a non-electrolyte per litre of water. Thus for example. Molar concentratio ns.lon and OsrnosLs 113 same. The solution is said to be hypertonic. In exerting a. In contrast to tonicity. When eryth rocytes mM mannitol no change in the size of the ceils is observed. If a substance iordzes. a non-lonizable has an osmolar value of 1. It is a function of the cell membrane. In quantifying the os motic of body fluids. Tonicity of a solution is there fore expressed always in terms of response of cells immersed in solutions. has one important disadvantage in being temperature dependent. i. This however. The extent of swelling depends on the osmotic pres sure differences between cell contents and the surrounding fluid. for example a tank divided which is permeable to solvent and the solute being completely impermeable to sucrose (see Figure 4. Chamber 1 is with an squeous solution containing 1.Figure 4. hyperosmotic and hyp0smotic are not synonymous to hypertonic and hypotonic. If we empty out the tank now and fill chamber 1 again with the same mixed of sucrose and glycine while chamber 2 now is filled with 1.0 M sucrose solution only . The above observations make it clear to us that/sosmotic is not synonymous to so tonic. 280 mM glycerol is hypotonic to the same cells.4 case 2) we observe no net transfer of water from any of the chambers .0 M sucrose and 1. the tonicity of the solution in chamber 1 is less than that of the solution in c hamber of this hypotonicity of the mixed solution. in . .the cells will swell. A solutio n isotonic can be slightly hyperosmotic to it and vice versa. Similarly solution has to be prepared at a concentration of 337 mM to be isotonic for red equal to 0.4 case 1).0 M glycine while chambe r 2 a 2. i. Thus knowledge of reflection coeiTcient can serve as during preparation of isotonic solutions for living cells.n the osmotic behaviour of living cells. Thus the pattern of sel ectivity of the plays a great role i. of the fact that the solution in chamber I was initially hyperosmotic to that in chamber 2. Consider.0 M solution of by osmosis. To ma ke glycerol a concentration of 318 mM is necessary to give an effective osmolarity !280 milllosmoles.e.88for glyce rol. water escapes to the 2. The reason for this is the value of G. Chamber I is thus isoosmotic with chamber 2 .83 for malonamide. This is. which is 0.0 M aqueous solution of sucrose. reason for this is the isotonicity of the two solutions. ' of a solution cannot be predicted solely from its known composition. 0 M Sucrose or n I CASE 2 ] (1) Chamber 1 is hyperosmotic to chamber 2.114 2.0 M Sucrose 1. (II) Chamber 1 and chamber 2 are isotonic to each other.0 M Glycine 1. Chamber 1 1.0 M Sucrose 1.0M lycine Biophysical Chemistry Chamber 2 Chamber 1 Chamber 2 Selectively permeable membrane : .0 M Sucrose 1. In determining the molecular weight of a protein. concentration of solute in moles/litre. For example. an impermeant molecule of s mall mass has the same effect as a molecule of large mass. the osmotic pressure-mol ecular weight relationship holds only for very dilute solutions. at least in the dilute solution limit where interaction effects are a voided. no osmosis. (II) Chamber 1 is hypotonic to chamber 2. V But as 'C' is equal to c/M . since th e van't Hoffs equation is followed for osmotic pressure measurements. since osmotic pressure is a colligati ve property and therefore depends on the number of solute molecules. etc. . charge and so for th of the solute particles. The van't H offs equation is used for the purpose in the following manner. i.permeable to glycine CASE i I but not to sucrose (I) Chamber 1 is isosmotic with chamber 2. plastics. Figure 4.4 Osmotic behaviour of cells MOLECULAR WEIGHT DETERMINATION FROM OSMOTIC PURE MEASUREMENTS Osmotic pressure measurements for molecular weight determination is seldom used today because of several disadvantages. proteins.. measurement of the osmotic pre ssure of solutions having different small concentrations of protein are made. l-IV = nRT As n = C.'.'.e. it is indifferent to the size. shape. direction of osmosis ===== . Therefore this method can be put to use in determining molecular weight of such high molec ular weight compounds as polymers. this method has impo rtant theoretical advantages. in practice. Secondly. Firstly. However. cRT MI-[ . R is the gas constant... +na . the osmotic pressure In atmospheres. niMi Run ffi n +n. each of a different mo lecular weight. T is the temperature In Kelvins and H.n moles of m olecular weight M. us moles of molecular Ms and so on. A graph ofl'[ /c is against c. n]M] + n2M2 + n3M3 +.. From this the molecular weight of th e proteIn (see Figure 4.5). Then..nl Is the number of moles In the l fraction of molecular weight Ml . The linear curve obtained is then extrapolated to zero concentration which the value of the Intercept as equ. Thus. i. to i 0 &l O 03 0.70 Concentration in gm protein/litre solution The osmotic pressure measurements give a value of M. the average weight of a mo lecule..oncentration is gms/lltre. the sample contains nI moles of molecular weight Mi.. Weight of the sample (w w M.. to RT/M.and Osmosis 115 ".4 05 (6 0. iis equal to the weight of the sample divided by the total number of moles In th e sample.e. = or number of moles Number of moles (n) M n Suppose n' of the sample is made up of several fractions. The 'number' term therefo re is replaced by 'Ight' of that species In the formula represented as : My = wlM +W2M +wsMs +"" wIMi . to various molecular s pecies In proportion to their given weights In the given sample. The weight average molecular weight gives representation.or Run . Measurement of any of the colligative properties of a sample will lead to the ca lculation of number average molecular weight as they are proportional only to the number of u nits In solution.'-niMi En Such an average molecular weight Is known as a number average mo/eadar weight. 12. 5. 2. 8. 3. 9. 14. 7. 13.1 enlists the molecular weight of certain proteins as deduced from measu rements of their osmotic pressure. No.116 or ffi hiMI. 12. 10.0 Table 4. 6. 4. . and so on. 0 36.0 69.0 580.45. Huma plasma L-Myosin 17.0 840.1 67.0 26.5 24. Name of Protein Insulin (in acid medium) Lysozyme {egg white) Trypsin inhibitor (Soyabean) Prolactln Trypsin Pepsin Chymotrypsinogen Chymotrypsin Ovalbumin Haemoglobin Bovine Serum Albumin Human Serum Albumin Flbrinogen.0 44. These primitive living things live d in an environment which was more or less constant with respect to the composit/on of v .9.5 36.0 41.0 BIGNIFICANC OF OBMO818 IN BIOLOGY Most of us believe that life began millions of years ago in the form of minute s imple organisms dwelling in an aquatic environment.Table 4. I Molecular weights of certain proteins from masuremnts of their omotio pr.0 69.5 36. ar/ous substances.3) .o wn 'personal' internal environment while the world surrounding them formed the e xternal environment (see Box 4. This process of evolution finally gifted the with theh. the organisms gradually evolved into more complex an d highly organ/zed individuals. As time passed. 117 Diffusion and Osmosis . Many physlological processes and most of the orga n systems therefore got involved in maintenance of this constancy of composition of the in ternal environment. which is also known as homeostasis. The body. This state is one of osmo tic equilibrium.environment became vital for existence of li fe. a dynamic equilibrium was achieved. The factors responsible for compartmentalization of body f luids appear to be largely osmotic and related to electrolyte concentration. It is at this point of evolution that the different exchange mechani sms for the to and fro passage of molecules in and out of cells got an upper hand. In other words. It had to be maintained at all costs. di vided into many many compartments by an effective network of membranes worked in a perfectly coordinated manner. Fluids containing dissolved matter were exchanged continuously between t he "internal" and 'external' compartments.118 BiophyslcaI Chemistry Gradually the constancy of internal. by an incessant replacement and exchange phenomenon. The body fluids distribute themselves acro ss membrane structures such a way as to equalize osmotic pressure. Osmotic pressure plays a major role in the maintenance of this dynamic equilibri um within an individual. . This is further mod ified by 'the selective nature of the biomembranes. pancreas and agaln intestine./Mffuslon and Osmosis 119 Greater the concentration of non-diffuslble particles on one side of a membrane as compared to the other side. lungs. These m echanisms are well adapted for exchange of water. The organs responsible for malntaining homeostasis in an individual are many.4). . i. liver. minerals and metatlotds extstLng as cations (see Box 4.e.. Bu t the homeostatic mechanisms or their end organs mainly lle in the intestine for absor ption and for exction in the kidney. skin. more Is movement of water towards It. towards high solute concentration. Though its value is quite less than the total osmotic pressure of blood.0 mm Hg whereas at the arteriole end it rises to 25. an appreciable variation in the concentraUon of c ertain electrolytes retained in body fluids can drastically affect a large number of ph ysiological functions.Besides the plasma proteins. it exerts a great influence in maintaining fluid bal ance across the capillaries. a normally negative interstiti al fluid pressure becomes positive. This may happen due to cumulative or singular effect of severa l factors like high blood pressure. a loss or deficiency of plasma albumin has a mor e negative effect on fluidbalance in the body. This force is enough to hold a certain volume of water within the blood. The opposite occurs at the venule end of the capillary with fluid flowing in fro m the tissues into the blood. are large bulky mole cules which cannot normally diffuse through the blood capillary membranes into the rel atively protein free tissue fluids. fluid accumulates in the interstitial space r esulting in a condition known as edema.0 mm Hg.Biophjsical Chemistry te/eost. being macromolecular polyelectrolytes. In this diseased state. However. At the venule end it is as low as 9. Albumin contributes to around 80°/0 of the total osmoUc pressure of the plasma pro teins. The total osmotic pressure of a body fluid is equal to the sum of the osmotic ef . 7. At the arteriole end additi onal outward forces like the interstitial.fluid pressure (approx. The colloidal osmotic pressure of the plasma proteins is opposed by the cap///ary pressure which tends to direct the fluids out of the capillary into the tissue.0 mm Hg) also exert their effect with the result that there is net fluid shift at this end from the capillary to interstit ial space.of approximately 25 mm Hg. Lowered plasma albumin leads to diminished b lood volume which precipitates into a state of haemorrhagic shock. r The plasma proteins. Because of its considerably low molecular weight compared to globulin.. it is a m ore effective species osmotically. increased capillary porosity and/or low plasma protein cont ent. . the co//o/da/osmotic pressu re (also called ondotic pressure) which has a magnitude. They thus exert an inward force. Therefore. probably due to the similar nature of their functions. if this balance between the two opposing forces is upse t under certain abnormal physiological conditions. This filtering force differs in its magnitude at either ends of a capillary. An increase in potassium concentration in blood mainly affects funcUoning of the heart and also causes dilatation of blood vessels.33 Min its total concentration of dissolved p -rarUcles inclusive of all lo ng andhon-electrolytes). blood plasma is 0. A major loss of sodium from the body leads to asignificant lowering of osmotic pressure of the body fluids which may be enough to cause dehydration whi le increased serum sodium leads to fluid retention. . potasslumand calcium. The three important cations which require special mention are sodium. The cationic components of blood require precise regula Uon in order to matntaln the osmoUc balance of blood.e . Decreased serum calcium may lead to a generalized muscle spasm called tetany. The osmolar concentration of plasma is approximately 0. The vascular system thus loses ittonicity.33 (i..fectiveness of all the long present. The Mathemat/cs of D/. Diffusion and Osmosis in Comprehensive Biochemistry (Florki n. Academic. M. shrinkage of the protoplasm from the walls is a regular phenomenon observed due to leakage 0fwater from the interior of the cell through the membrane to the exterior when cells are placed in hypertonic medium. However. plasmolysis. 4. Vol. eds.). Glasstone..... Robinson. Van Nostrand. swelli ng of the cell in not observed. When thes e healthy cells are placed in. 1975. Textbook of Physical Chem/stry. i. R. the cells are called 'turgid'. J. 1962. 5. W. Stein.e.solutions of low and high osmotic pressure respectively the cells eith er swell or shrink. Edsall.. Elsevier.H. Crank. 2nd ed. The constant osmosis of water into the cell creates a surplus hydrostatic pressure w hich keeps the cell strong and elastic. But due to the rigidity of the outer cellulose walls. 1959. New York. i. Vol.H. . Suggestions for Further Reading I... eds. E. 3.T..).ctrolyteSolutions. New York.D. Butterworths.Bailey. 1B (H. J. S. and Stotz. 2. New York. Oxford University Press. E. and Stokes.A.rusion.and Osmosis 121 Osmotic pressure has a key role to play in the functioning of normal healthy pla nts. The Size.Diffusion . In this state.. plasmoptysis. Neurath and K. London. 2.e.. 1953. R. Shape and Hydration of Proteins in The Proteins. 1956. Stationary plane Force Moving plane Stationary plane Figure 5. .. I Velocity gradient in a fluld flowlng through a glass tube In a liquid sample. .1). so formed can be related to the shearing force applied per unit area b y the equation.oil are allowed to flow out through I0 ml glass pipettes under identical conditions. Th is difference in the rate of flow of liquids is attributed to the phenomenon of viscosity. In the following text we shall elaborate this term to understand its nature and impact on the living syst em. viscosity of the liquid. The velocity gradient. layers. dv =dx difference in velocity between two layers separated by a distance dx. Each layer will move with a different velocity (see Figu re 5. This means that one liquid flows faster than the other. a layer of liquid experiences resistance when it flows over another layer. we observe a time difference between the tw o liquids in the emptying out process. If a shearing forceI is applied to overcome the attractive forces between the molecules of the adJacenta. F dv n = q {I) A dx "'" F where = shearing force per unit area (also called shearing stress).5 VISCOSITY If equal volumes of water and castor . Such an apl]led force is called a shearing force.S Force : If a piece of metal is subjected to a force at its upper surface while being held fixed at its lower surface. it will be deformed. . The deformation is proportional to the a pplied force. The units of viscosity are dynes sec. can be defined from the above equation as the f orce per unit required to maintain unit difference of velocity between two parallel liquid sur faces which unit distance apart. let us consider the viscosity of a solvent lo. The concentration 'C' is expressed in grams/ml or grams/100 ml. Many empirical equations have been proposed to describe the change of specific viscosity with c oncentration but the equation proposed by Huggins has found wide acceptance. our main concern in this chapter will r viscosity with an eye on the solutions of macromolecules.poise). the specific visc osity also increases. More common uni ts are the poise) and millipoise (10... On the other hand the e high of viscosity and do not flow easily. Therefore.cm-2. after the name of poiseuflle who p ioneered r of viscosity. As the concentration of the macromolecules in the solution increases. The ratio of the change in viscosity to the original viscosity of the solvent is called the specific viscosity and is de noted by qsp. we assume that the fluid concerned is undergoing a lam inar the flow lines of the solvent will cause a change in the viscosity of the solvent. Most of our body fluids the whereas viscosity i low coefficients of iscous' liquids hav are viscous due to of particles of macromolecular size. It is expressed as . On addition of macromolecular particles to this solvent.between tw o parallel one cm apart and have an area of contact equal to one sq. l. a liquid is said to have a coefficient of vi scosity as one poise a force of one dyne maintains a difference of velocity of one cm sec. Therefore. {2) The term fluidity expresses the tendency of a liquid to flow s a measure !the resistance a liquid offers to this flow. When we define viscosity.123 C=O The coefficient of viscosity. The ratio of solution to solvent viscosity (q/qo) is the relative viscosity. Liquids having viscosity are 'mobile' due to their ease in flowing. the viscosity of the solvent increases to a new value q. The reciprocal of the coefficient of viscosity is called the fluidity and is giv en by the symbol 1 =- . Now.cm. depicted by [q].2 Plot of reduced viscosty versus concentmt .A plot of q. On extrapolating the curve to zero concentration.p/C verses C is shown in Figure 5. The value oflp/C at this dilution gives us the b-ttrtns/c viscosity of a solutio n. The Huggins constant 'k' in the equat ion (3) is obtained from the slope of the curve. we get an intercept on the Y-axls. Concentration lure 5. We also call it the//m/ring vlscos/ty number.2. Such plots are normally linear for dilute solutions.plC is sometimes called the reduced v iscosity. The quantity . (5) where v = the viscosity increment which increases with increasing axial ratio p ffi l/d (the ratio of length to diameter) of the macromolecule and $ = fraction of the volume of th e solution occupied by the particles. Nevertheless. The equation is stated as where In I = In (1+ l) When macromolecular particles are suspended in a solvent they assume different conformations.. The equation 1sp-.. Scientists have made several attempts to quantitatively r elate viscosity to the axial ratio of t-he macromolecules as . (6) is based on the following three assumptions : (a) the solute particles have a Very large size ratio to the solvent molecules a . The value of [B] determined by this method is only mo derately accurate because it requires the measurement of.124 .p= v$ ...5 $ .. It is necessary for determination of viscosity for those solutions whose protein components denature easily when a very dilute solution of the protein is allowed to flow th rough a capillary.2.. The increase in viscosity then depends primarily on the effective volume occupied by the macromolecules. [i) Sphe Einste/n gave an equation relating spec/flc viscosity of a di/ute soluti on of macromolecules to the volume fraction occupied by the particles. (4) . another equation proposed by Solomon and Cluta has been used extensively.p at a single concentration only .. (9) For moderately concentrated solutions.. equation (8} gives "q. 1sp = 2. {8) M where We is the effective hydrodynamic volume of one mole of macromolecular solu te particles of molecular weight 'M' and concentration 'C' gm/ml.6// C C M As the concentration reaches Lnflnite dilution-. so t hat the. equation now takes the form. |'q) = 2.-.6 As the volume fraction $ VeC = . When the solution is moderately concentrated.5 $ ÷ 12.5 V/M .nd are rigid. (b) the flow rates are very low.5V= + -.12. another term is addedto equation (7). there is interaction between the f low lines of the solute too and to quantify it... and (c) the capillary through which the liquid flows must have a large radius co mpared to the radius of the spherical particle. the equation becomes. = 2. 3). AS O. sp p2 2 or v p = 14 + (11) 15(In2p. Prolate {Approx. (12) a<<b factor {V}. For oblate ellipsoids..1:5) 5(ln2p-0. According to derivations by Einstein and later by R. The prolate e//Ipsolds of revolution are clgar-shaped while the oblate ellipsoids of revolut ion are dlsc-shaped (see Figure 5. 108 %) . t he equation takes the form.5)' 15 "'" The above equation is applicable to prolate ellipsoids.. (p) 15 tanp O Prolate O Oblate spheroidal spheroidal (Approx.revolution. 165 %} 125 Ellipsoids Simha provided an equation for the viscosities of ellipsoids of.. Slmha. (Approx.3 Ellpsolds of revolutlon Oblate (Approx.great deal of rotation about the various segmen ts in the .4 Plot of Simha" s viscosity increment v as a functWn of axlal ratio (a / b) for prolate and oblate ellpsokls of revolution (til) Linear Random Coils No particular three dimensional structure can be allotted to a linear random col l. This is because the molecule exhibits a .%) a>> b {a} 0 Prolate Oblate {d) Figure 5. 94. 63 %) Figure 5.4 shows a graph of the axial ratio for ellipsoids of revolution against Simha's 15 Oblate "o lC Prolate 1 25 5 I0 15 20 25 Simha's factor Figure 5. Myoglobin Tropomyosin (a)" Serum albumin [horse) Serum albumin 0roman} y-Globulin (human) Flbrlnogen (human} Thyroglobulin . viscosity increments.126 Bfphysfcal Chemistry macromolecular chain. The intrinsic viscosity for such soluUons becom es a measure of the particle volume. Table 5. The r andomly cotled macromolecules occupy a very large effective volume in solution. 5. 3. Table 5.1 Itsts the values of intrinsic viscosities. 9. 4.1 Intrlas/e viseoIt/es. only average dmenslons of the molecule can be deted. vlscosRy increments and axi al ratio for a number of proteins. For the same reason. No. Hence. 10. the intrnsic vscosltles of solutions of such polymers are often found to be vast ly larger tha those for spheres or ellipsoids. II. and calculated axial ratio s for various St. a large number of conformations are possible. Name of Prote/n I00 [] 2. 6. As the conformation of the molecule continuously changes by the npacts of the surrounding solvent molecules. 0 .52 6.0 4.0 25 7.6 4.1 3.2 5.3 .0 3.1 28 5.9 39 Axial Ratio Prolate Axial Ratio Oblate w = 20.9 4.6 3.4 3.2 4.2 6.3 3.6 8.Tooacco mosaic virus 3.0 5.1 35 9.3 28 24 6.7 74 8.0 2.1 w = 0.5 4.1 4. 1 8.0 5.9 18 16 w=0.6 4.3 6.5 2.3 5.3 4.8 6.3 6.5 5.1 w=0.0 8.3 5. The values expres%-d .5 15 7.4 4.4 56 4O The values of tropomyosin vary with different conditions of the experiment.3 3.9 3.4.8 44 26 11.1 5.2 3.0 17.0 94 76 8.5 4. : if 30% then w = 0. 2. It is a measure of the three dimensional ex tension of a particle. I.here are for acid Solutions at pH 2. When the molecule is a linear chain. intrinsic viscosity. gives a rough measure of the effective radius of a m olecule (see Box 5.. w = O. v' = viscosity increment w = degree of hydration.e. i. Rod/J ofGyratlon : It is the square root of the mean square distance of all individual electrons from the centre of gravity of a particle. .3 and so on. I.1). The radius of gyratOrS. the radius of gyration will be a function of the chain length of the polymer and hence of the molecular weight. if hydration is to the extent of I0%. Rg. 127 . . (13) where K and a are constants for a particular solute-solvent system and temperatu re. the molecules in the liquid become more active.. It depends on the number of bonds in a chain and takes account of the expansion of the flexible coil due to the continuous bombardment of the solvent molecules on it. and the liquid now flows easily or we ma. say the liquid has become more mobile.. If the polymer is dissolved in a 0-s olvent. The maximum value Of th e exponent a in 'good' solvents is 1. If the flexible polymer is placed in a "better" solvent where there iS much inte raction between solute and solvent. and M is the molecular weight.0. where Mo isthe Now. FACTORS AFFECTING VISCOSITY Temperature When a liquid is heated. a is also known as an expansion factor. The variation of the viscosity of a liquid with temperature is best expressed by the equation. This in creased molecular activity or molecular motion occurs at the expense of cohesive forces acting between the molecules. . the liquid now faces lesser resistance to its flow. We observe a decrease in viscosity. (see Chapter 3) the intrinsic viscosity [I]0 becomes [1] = KILL' (14) as a = I/2 in such a solvent. then the radius of gyration will increase more rapid ly with M. the intrinsic viscosity of a solution of macromolecuIes is related to the m olecular weight by an equation stated by Mark Houwink as (I) = KILL . As a is dependent on molecular weight. it will also increase. As a result.Biophysical Chemistry (ii) For rods. The parameters K and a are are determined by measurem ents of [I] for each s01ute-solvent system using for calibration samples of known molecular weig ht. and E = the activation energy per mole. {16) or log I = T where A = constant depending...upon the molecular weight and molar volume of the l iquid. ..I = Aesj .. T = temperature of the liquid. (15) A+E . 9:3. °C 60 40 20 0 -1. This is found to be true for a large nu mber Figure 5.e. the plot of log l against the reciprocal of the abso lute i. 57°C 60oc Temp.5)...1. with an increase in temperature. should yield a straight line. The molecules thus require a higher amount of activation energy to the resistance and increase flow.7 -1.4 :3.erature Flgue 5.:3. and hence viscosity decreases in a reciprocal manner.7 103/T lure 5.5:3. number of molecules which acquire sufficient energy (activation energy) to take part in [ flow increases. :3.9 2. T The opposite is seen in gases. This increase in the number of molecules is in proportion to t he Boltzmann e-E1er. The explanation offered for such a plot is that astemperature incre ases. For liquids having many hydrogen bonds some of the energy obtained will be utili zed in the H-bonds. As a result.5 Temperature de1rtence of vscost plotted as log r vs.0:3.6 Plot of log of tlme of JIow through vlscometer of an albumin solution as a function of temperature. i..:3:3. The temperature effects on viscosity of . viscosity of such solutions is al ways temperature.Stage B 129 According to equation (16). l/T. viscosity rises.6 :3.e. The other lyophilic .by Ostwald (see Figure 5. It seems as though just before coagulation. upto a certain stage (stage A) after which there is a sudden shoot up and an equally sudden fall in viscosity of the albumin solution (stage B). After crossing this fluctuating stage the viscosity curve appears even (stage C) and as if in continuation to stage A..lyophilic colloidal solutions are very queer.6). The viscosity of albumin solution with changes in temperature have been studied in detail . As the temperature is raised. albumin increases its degree of hydration and as coagulation is completed it is left bereft of water. the viscosity of the solution decreases. The coagulated albumin is hydrophobic in nature. (Fi . As per equation (17).. The effect of pH on viscosity of colloidal solutions is very distinct. This increase in viscosit y arising from the movement of the ionic atmosphere when the charged molecules move is called the e lectrovscous effect.Fr ) means the potential difference between the inte rior of the particle and the interior of the liquid. However. o = viscosity of the medium. r = radius of the spherical macromolecule.5 1+ 22 j j . where k = specific conductance of the surrounding medium. for practical purposes it is a ssumed that (FI . The charges produce electrostatic effects which can profoundly influence the sol ution behaviour of the macromolecule. Fr = potential difference between the surface and centre of the particle. The macro molecules contain a nurn'tmr of positlveand/or negative charges on them (therefore called polyelectrolytes). The charges on the molecules induce opposite charges in the surrounding solvent.Blophysa Chemistry systems like those of starch and gelatin also show such anomalous behaviour thou gh they may be quite different from that shown by albumin. Kras ny-Ergen derived an equation for the electroviscous effect.. The electrical double layer which is thus formed moves along with the particles resu lting in an increase in friction thereby increasing the viscosity.Fr ) is equal to the potential difference across the double layer and the term (zeta . Fi = potential difference between the surface of the particle and a point in the medium where the charge density is zero. D = dielectric constant of the medium. It is affected by changes in pH and ionic strength of the solution. It is written as 3 1 D r)r = I+2. 1 to 0:2 M sodium chloride solution. This is due to the fact that the molecules of this type lack fl exibility in the native state. there are some exceptions to this rule. there is either an excess of negative charges or positive charges and the molecule expands. But. This will keep th ionic atmosphere around the macromolecule unchanged during the dilution process. If the polyelectrolyte expansion. Above or below this pH. for example ribonuclease. This leads to an increase in the visc osity. When working with proteins. At the isoelectric pH [the pH at which the net charge on the molecule is zero). the electroviscous effect is minimized by c out visc osity measurements in 0.potential] replaces it. the electrostatic forces of attraction within the molecule cause it to contract resulting in a dec rease in the -viscosity of the solution. Globular proteins. This greatly reduces the extent of the electric double layer and hence leads to reduction of the electroviscous effect. the dilution of the macr omolecul can be done with a solution of the same ionic strength (isoionic dilutionl. d o not show a appreciable increase in viscosity even when they have attained their maximum pos itive or negative charge. has to be controlled. . This means that as pressure increases. elongated molecules show a high viscosity.(19) The equation shows that as specific volume increases. they form aggregates and the.Batschinski in 1913 ave an equation relating viscosity to the specific volume of" a liquid as C - .. viscosity of the liquid also increases.. When the solute molecules interact strongly with one another.. Chemical Compotion The viscosity of liquids generally depends upon the size. viscosity decreases and vi ce versa. v = specific volume of a liquid and b and c are constants. Specific Volume . (0 capillary flow. The degree of orientatio n ofa particle is dependent on these factoi's. v = b+-. and (//0 the rate of fall of a ball through solution.. viscosity of the solution increases. . Liquids with large. Large amounts of dissolved Solids also incr ease viscosity. (i0 rotation of a cylinder immersed in the solution. The viscosity measurement employs the use of atleast three different instruments based. shape. This elationship is applicable only over a range of temperature. On rearranging Eqn (18) we get. (18) v-b where vl = viscosity.131 Viscosity bears a linear relationship with pressure in the case ofllqulds. flexibility and rotational diffusion coefficient (see Chapter 9) of the particles.J. I = length of the capillary in cn . V = volume of the liquid in milliliters..8 IV. method is based on PoLse ug/e's Law which embodied in the equation .. R = radius of the eapillary in cm. This. cm. (20) where = viscosity of the liquid. P = pressure applied in dynes per sq.The time required when a liquid flows through a capillary under specified condit ions can be used to determine the viscosity of the liquid.. t = time of flow in seconds. once the densities of the two liquids are known..i (21) . A glass capillary viscometer can serve the purpose. This permits us to ca lculate the viscosity of the unknown liquid i provided of the reference liquid is known. I can be made constant by using a capillary of known dimensions for all the liquids. by capillary viscometer the viscosity of one liquid can be determined by m easurement of its flow relative to another liquid whose viscosity is known.. the pressure under which the liquid flows. {22) 132 Biophysical Chemistry The Poiseuille's Law states that the volume of a liquid flowing through a capill ary is directly proportional to. . Also. Design of a Viseometer The Hess viscometer is often used to measure viscosity of blood. the volume of liquid that flows is inversely prop ortional to the length of the capillary and to the viscosity of the liquid. V.. The values R. and t he fourth power of the capillary radius. Thus. The value /p is also known as the k/nernat/c vlscostty of the liquid. Wat er is generally used as the reference liquid. If we measure t he time of flow of two different liquids through the same viscometer. the viscometer is calibra ted for the measurement of the flow time tt and t2 of the two liquids. the flow time.2). then according to Poiseuille equation. It utilizes hor izontal capillaries instead of the vertical ones usually found in other capillary viscom eters (see Box 5.. the ratio of the viscosity coefficients of the two liquids is given by Since the pressure P and P2 are proportional to the densities of the two liquids p and Q2 we may write r12 t2P2 tp Thus. For thesellquids.7 A Couette rotatlng-cyllnder viscometer where T = r. the non-Newtonian liquids-display a change in viscosity with variation in the shearing stress. The c onstants of the viscometer appearing in the equation can be determined by accurate measur ements. On the other hand.the Solution When a rotating body is immersed in a liquid it experiences a resistance in its movement. The type of flow obtained in this type of coaxial rotating cylinder viscometer is simpler th an that in a capillary viscometer. The amount of retardation dep ends on two factors: (/) the viscosity of the liquid. Liquids or solutions for which the viscosity is constant and is not affected by the shearing stress are called Newtonian liquids.trolllng the temperatureof the system. (23) Fre 5. whose viscosities are known can be used for calibrat ion. Alternatively. and . two liquids. Non-NeWtonian behaviour is shown by molecules which are very dI Rotation of a Cylinder Immersed in.Wscoslty 133 Torsion . l can be calculated by equation (23). the thermal motion of the molecules is capable enough to counteract any effects due to flow itself and the liquid thus exhibits Newtonian ilow. The only difficulty lles in con... = h = Once the values ofT and 0 are known. This is due to the viscosity effect of the liquid. radius of inneT cylinder. -.P°tnter %'nple The above principle was utilized by Couette in. r2 r2 . radius of outer cylinder. the liquid rotating in the outer cylinder causes a torque. The inner cylinder is held by a torsion wire (see Figure 5. This torque and the angular velocity of the rotating cylinder is recorded.p.7). The viscosity of the solution is related to the torque produced by the equation. height of cylinder immersed in the liquid.I Circular 't-. =T o torque required to maintain a constant angular velocity. angular velocity of the rotating cylinder. .() the speed of rotation of the body. .1890 while designing a viscometer to measure the viscosity of high molecular weight systems. The apparatus consists of a rotating cylindrical cu. In the Couette rotating cylinder viscometer. P) g = 6 r 2r (P-p)g or q = . Th e capillary viscometers produce a very high shearing stress and are therefore unsuitable for viscosity measurements of DNA solutions. 4/3 r (0 . Let us consider the particle to be a sphere of radius 'r' and density p.6 When these two opposing forces just balance each other. This constant velocity is attained du e to two opposing forces acting on the particle at the same time. They are (/) the centrifugal for ce acting downwards to accelerate the body.pL) g . As the Couette viscometer tends to minimize the n on-Newtonian behaviour of solutions. as stea dy state is reached and the body now continues to fall with a constant velocity. The liq uid through which it fails has a density PL" The gravitational force acting on the particle pulling it downwards is given by F = 4/3 r3 (p . (25) At equilibrium. A particle whose volume is much greater than the molecules of t he solvent falls through a liquid with a constant velocity. and (//) the frictional force acting in the opposite dir ection which causes an upward drag.. Rate of Fail of a Ball Through a Solution The falling ball viscometers are designed to measure viscosity of solutions by a pplying Stoke's formula. F. (26} . balances F2 so that. The frictional force acting simultan eously on the particle leading to a visco .us drag can be given by F2 = 6 r l v ..... it can be efficiently used for viscosity measurements of DNA solutions also..134 Biophysical Chemistry asymmetric or extremely flexible or are in the form of stiff coils as in DNA. (24) where g is the acceleration due to gravity. The same procedure is followed replacing the unknown liquid by a reference liquid whose density and viscosity are known. Inlet -. provided the radius of the falling body 'r" is lar ge compared to the size of the solvent molecule. the body tends to "drop" instead of failing with a constant velocity.8 The falllng-ball vtscometer . and the time of fall of the ball between two marks is measured.Sample A small steel ball of density p is dropped through the neck of a cylindrical tube (see Figure 5.9v Equation (26) is called the Stoke's equation and is applicable to the fall of sp herical bodies in all types of liquid media.-Steel bali ] : -. The viscosity of the unknown liquid can now be determined by the relative time of flow of the two liquids as given by the equation Figure 5.-.8) filled with the liquid whose viscosity is to be determined. If the difference is not large. I2 = density of the reference liquid. plotting a graph of specific viscosity versus shearing stress.000 daltons. 12 -. which generally exhibit nonNewtonian behaviour. p = density of the steel sphere. For viscosity measurements of macromolecular solutions.. which show pronounced non-Newtonian behaviour. However. t = time of flow of the reference liquid.al different conce ntrations. The method which is gener ally followed is of extrapolation to zero rate of shear.. We can thus find the intrinsic viscosity of the macromolecular solution at . t = time of flow of the unknown liquid. the former is to be pr eferred. l = density of the unknown liquid. The procedure for such a measurement is as follows : A concentration is chosen at which the specific viscosity of the macromolecular solution is measured at differing known values of shear stresses. for asymmetric macromolecules with a molecular weigh t exceeding 400. but in this case. (27) Viscosity 135 where -. with concentration denoting the X-axls in a manner similar to the Huggin's plot (see Figure 5. The graph is then extrapolated to zero shearing stress. Continuous measurements for a length of time under a specified shearing stress can also be performed. extrapolation to zero shearing stress is necessary to measure the viscosity. In this instrumen t viscosity measurements of macromolecular solutions can be attempted over a wide range of s hearing stress. the Couette viscometer is routinely used.viscosity of the unknown liquid.viscosity of the reference liquid..2). The value of (Isp) = 0 ('T' represents the shearing stress) so obtained is plotted along with values obtained similarly by repeating the procedure at sever. This is to be followed by. If the maximum shearing stress is denoted by. For the mac romolecular solutions whose intrinsic viscosity can be measured using the Couette viscometer operated at varying shearing stresses. The equations given below for maximum shea ring stress and maximum rate of shear can be used for calculating the value of the various s hearing stresses applied which are in fact. equation (30) can be applied. It is a multigradlent capillary viscometer which ca n accommodate particular ranges of shearing stress.zero shearing stress. . = (28) 2L "'" 4V Rm = (29) where V is the volume of the liquid passing through the. capillary of . . necessary for the extrapolation.radius 'r ' length 'L' in 't' seconds under a pressure amounting to P dynes per square cm. The apparatus which can be used for the above experiment is a modified t ype of capillary visoometer proposed by Yang.? and if Rm represents the maximum rate of shear. 2% solution of myosin had observed a viscosity ratio of 1 . T hey are particularly useful in a showing structural changes in a macr. The combination of viscosity with data ob tained by other techniques like viscosity and radius of gyration(obtained from light scattering) . rd is the difference in radius of the two cylinders of the Couette viscometer an d h is the height of the cylinder'immersed in the solution.. (30) Blphysaxl Cherrdstnj where 7 is the shearing stress. and/or ( ) associationdissociation behaviour of subtmits of macromolecules. Determation of Aymmetry. Thus their behaviour i s non-Newtonlan. Edsa ll and Mehl (I 940] working with 0. caution is required in interpreting the obtained data. APPLICATIONS 0' VIOMETRY Viscometry has found wide application in the study of macromolecular properties. The intrinsic viscosity of such a solution will decrease with increasing shearing stress. for proteins. flexibility. and more so. The degree of orientation of a particle depends on its size. T is the torque applied to maintain a constant a ngular velocity. and rotational diffusion coefficient (see Chapter 9]. degree of asymmetry of the myosin molecule.omolec. But in many cases viscosity measurements alone provide definitive information. Siza md Shal of Maeromol¢lu Asymmetric particles do not rotate evenly in a velocity gradient and tend to ori ent themselves in the direction of the streamline of flow of the liquid. e associat ed with physical or chemical changes in the environment such as (0 protein denaturation. can give us Information about the shape of the molecule. shape.. This indicated a very . In the following text we s hall discuss in detail the relationship of some of these properties of proteins with viscometry.6 and a viscosity increment (intrinsic viscosity divided by partial specific volume) of 340 at hig h shear rates. Though among all physical techniques viscosity measuremen ts are simple. . since with increasing stress the degree of orientation of the molecules will inc rease. More often viscometry is combined with other physical techniques to obtain fool-proof information about the macromolecu le being studied.. The rod-llke proteins have a high axial ratio and give rise to very hig h viscosity increments because they tend to take up positions with the larger dimension oriented in the direction of current. The molecules thus lose their randomness. To obtain molecular welghts of macromolecules. In the 0 solvent a is equal to 0. the degree of orientation increases as the shear stre ss increases. 5 whereas for. In this position. The flexible ra ndom coils have an a value falling in the range 0. semi-rigid rods the value of a should range between 1.5 f or random coils. For non-solvated rigid molecules of spherical shape. a 1. Mark and Houwlnk on the basis of theoretical considerations modlfiedthe a bove equation and gave a better expression as [] = KMa The empirical constant a is dependent on the shape and solvation of the macromol ecule.5 to 0.8. the intrinsic vlscosity data may be combined with those of diffusion or sedimentatiom The Schachman equations can be appropri ately used in such situations which are stated as .0 and 1.5. For rigid rods. Staudinger (1932) on the basis of experimental considerations had given an empir ical equation relating viscosity to the molecular weight of a macromolecular solute a s [l = KILL Later. a = 0.The spherical molecules are compact and therefore give rise to relatively small viscosity effects. for e. there is an observed increase in viscosity.58xI0-s and M = where Szo Is the sedimentation coeffic|ent in Svedberg units at a temperature of 9. To obtain a detailed account of the denaturation process other techniques have to be combined with viscometry. [denaturing agent) shows a decrease in viscosity number. It is genera lly accompanied by a drastic change in one or more of the properties of the protein molecule. V is the partial specific volume of the subs tance (the volume increment when I gram of dry substance is added to a large volume of solvent) D= 0 is the diffusion coeiTcient at temperature 20"C.. (31) Viscosity 137 4690 ($2o) [rl]/ (i-%) 6.0"C. Protein Denaturation Denaturation is an alteration of the native structure of a protein. The refore. At 40"C there is an abrup t viscosity change indicating a partial uncoiling of the compact globular form that exists at lower temperature into the flexible at higher temperatures.g. It is more often an unfolding process. DenaturatiOn can be brought about by a vari ety of agents like acids. ultrasonic waves. This can be illustrated by the actio n of heat on the enzyme ribonuclease (see Figure 5. In contrast. alkalies. . viscosity measurements on proteins are useful In indicating changes in molecular configuration due to denaturation.. When a rigid molecule of low axial ratio is unfolded into f lexible chains. UV irradiation. heat. The intrinsic viscosity is sensitive to changes in macromolecular structure. a globular form might change to a random coil confo rmation thus Increasing the disorder of a system.9). myosin when treated with guanidine hydrochl oride . It is said that a dec rease in viscosity when rigid rods are converted into flexible coils. etc. Water is used as a solvent for this purpose... after measuring the [rl] of a series of proteins in 6 M guanidin e hydrochloHde have shown that an empirical correlation between [r] and the number of amino aci d residues written in the form .al..3 20oC 30oc 40oc 50oC 60oc 70°C Temperature Figure 5.9 Vscosity changes by the action of heat on the enzyme ribonuclease Tanford et. should not be taken at face value. Same can be said about the br eaking of disulphide linkages in a flexible coil which may lead to molecular extension. These conventions. higher aggregates are produced by the shearing stress in the viscometer. This can also tell us whether different denaturants acting on a protein produce molecules differing in shape or properties. the vis cosity of the solution should increase.. (33) where 'n' is the number of amino acid residues in the chain.138 Biophysical Chemistry [T]] = 0.000 residues. From th is. it should also decrease the solution viscosity.716 n°. Association-Dissociation Studies Association (aggregation) or dissociation of proteins can be studied by viscosit y measurements... On the other hand. ho wever. however. If the modification is of t he covalent type where some covalent bonds are broken or formed accompanied by a change in t he shape of the protein molecule. Thus. Other techniques like sedimentation and light scattering. By convention. association of myosin increases the intrinsic viscosity giving the indication that myosin association occurs end to end. it is very clear that viscosity is not very useful in association-dissociation studies and is used as an adjunct technique to absolute methods such as osmometry and light scattering. Chemicai Modification Most proteins can be modified by drastic treatments. the viscosity change is appreciable. To cite an example. Thus measurement o f intrinsic viscosity can also help us in detecting chemlcal modification of a protein molec ule. since side-by-side association dec reases the asymmetry. This is so because such an association is thought to i ncrease the asymmetry of the molecule. clearly show that myosin associatio n is side -toside. The above equation is applicable to protein molecules of all sizes upto 3. viscosity measurements of solutions that convert compact proteins to the random cot configuration can be related to the size of the polypeptide chain. if rod like molecules associate end to end. The 'increase' in viscosity in this experiment can be attributed to an art ifact whereby. Splitting of a peptide bond in a polypeptide chain may result in unfolding of a compact struc ture resulting in a considerable increase in intrinsic viscosity. .ss . Since viscosity is not dependent upon molecular weight of unsolvated spherical p articles. if dissociation of globular proteins involves considerable unfolding producing a random cot.10). Today we know that this polymer is the actomyosin filam ent that primarily constitutes the contractile apparatus of the muscle. Studies About Interaction Between Actin and Myosin Today we understand the mechanism of muscle contraction and the role of actin an d myosin in this intriguing phenomenon. However. Viscosity can be useful for understanding such types of dissociations. They further obse rved that addition of ATP to this mixture caused the viscosity to drop to the original level (Figur e 5. barely four decades back we were in dark about this mechanism. The scientists explained the results in the following way . I0). a considerable increase in the intrinsic viscosity can be observed. it is of limited use in association-dissociation studies of essentially spherica l particles. Given below are a few examples based on the discussion above. Albert Szent Gyorgyi and coworkers mixed purified actin and myosin in concentrat ed salt soultion.these two proteins must be interacting to generate large polymers. They observed that this mixing resulted in a large increase in the vis cosity of the solution (Figure 5. At this time important insight into the relationship betwe en actln and myosin and the relationship of ATP with these two proteins was obtained with the help of scosity studies. However. The inference was straight. I I).8 1.4 0. . .-TP.0 F-actin 1 0.5 mM ATP . Addition of ATP makes the viscosity drop.the previous experiment stated above.6 0.from striatedrabbit muscle. I actin myosin mixtures after addition of A TP. Effect of A TP on the viscosity of actomyosin in which the actin was derlved from the $11me mold and the myosin.pure actin from slime mold.5 mM ATP 0 I0 20 30 40 Time (min) Figure 5. solid line . I I. In purified state the so lution of proteins showed low specific viscosity.--. the viscosity dropped of.0. Moreover..4 0.2 0 Myosin When actin and myosin are mixed the viscosity of the solution rises. das hed llne .2 0. shot up (Figure 5.myosin from rabbit striated muscle.8 0. This means that actin and myosin from removed species still interacted to give rise to actomyosin. All they did was mix actin derived from the sl ime (a myxomycete) with myosin from striated rabbit muscle. Dotted line .mixture of acttn and myosin. |O)Actin-myosin mixtures.139 2.6 0.0" 0 Actin + Myosin ----> Actomyosin Actomyosin + ATP ----->Actin + Myosin 0 0. that Actin from Different Species Acts Sim/larly In an extremely interesting experiment Oosawa nd his colleagues proved that acti n from species indeed can act similarly. The moment these two proteins were mixed .to" 5 mM-A. when ATP was added.# 0. . the same effect would follow with guanidinium hydrochloride treatme nt also.9). and if one uses iodoacetate in the reaction mixture one can prevent these bonds from reforming. In the denatured condition the protein assumes a completely random coil configuration. However. much change in viscosity if they are treated with 6 M guanidinium chloride. Such proteinh will not give. Thus. In such cases. the same polypeptide wi ll experience a decrease in its compactness and in the presence of 6 M guanidinium chloride it will assume a random coil formation. On the contrary. if upon incuba tion i-n 6 M guanidinlum chloride. if consequent to such a chemical treatment. one assumpti on is that f the protein may be fibrous in nature and that denaturation has given it more fle xibility as a ( random coil. The same is true about the protein collagen also. if such bonds are broken. Detection of Intrastrand Disulfide Bonds in Proteins Assessment of viscosity can easily lead one to detect whether or not a given pro tein possesses intrastrand disulfide bonds. The viscosity \ of this polypeptide solution actually decreases upon denaturation meaning that t his polypeptide exists as a rigid rod in its native condition and the random coil conformation u pon denaturation is the more flexible form. An example is the enzyme ribonuclease (Figure 5. the viscosity actually decreases when they are treated with 6 M guanidinium chloride. Thus. one can safely infer that the protein was quite compact in its native conformation. the polypeptide will n aturally be present in a more compact shape owing to the folding that will result through su ch covalent bonds. Although in that figure the enzyme was denatured by i ncreasing the temperature. It could be that the prote in contains several polypeptide subunits that have dissociated upon guanidintum chloride tre atment and that this is the real cause of decrease in viscosity. However. one finds the viscosity of the . one sees an increase in viscosity of the protein solution. such a conclusion may not be always correct. [-mercapt)ethanol can reduce the disulfide bridges into individual sulfhydryl gr oups. it is observed that with some proteins. The viscosity will increase.140 Biophysical Chemistry Idea about the Native Structure of a Protein Incubation of a protein in 6 M guanidum chloride denatures it. An example of such a case is afforded by poly(/-benzyl-L-glutamate) . When such bonds are present. a short wh ile later. an increase in viscosity can be demonstrated. If no change in viscosity occurs. The logic is simple. Polymerization of DNA Later.protein solution increasing. This increase will not take place if one of the . if DNA. When this occurs. After this treatment though. The compactness of a given polypeptide will differ depending upon which cysteins are involved in disulfide bond formation.0 ml/gm. DNA polymerization can be studied without the use of radio activity also. When polymerization takes place. If the cysteins involved in disulfide link age are situated wide apart in the polypeptide.0 ml/gm when it has not been treated with [-mercaptoethanol. it means that the protein. The example there involves incorporation of radioactiv ity in the growing chain of DNA. in all probability. Thus calf brain tubulin has [1] = 36. a great change in viscosity upon mercaptoethanol treatment would mean that the polypeptide has disulfide bridges formed between distant cysteins. all four 5'triphosphate nucleotides. you will come across an example to study DNA polymerization using DNA. the molecule will be more compact as compared to the molecule which has disulfide linkage between two cysteins situated very near each other i n the polypeptide backbone. the viscosity of the reaction mixture must increase. it may mean the absence of such bonds. and Mg2÷ are incubated with DNA polymerase I. has disulfide bridges . This is a sure indication that disuffide bridges are prese nt in this protein. Thus. the chain length of DNA in creases. while a s mall ar indistinguishable change in viscosity would mean the presence of a disulfide bri dge between cysteins that are more or less adjacent on the polypepttde backbone. [1] climbs to 44. Thus. in the chapter on radioactivity. . the dye must get inside the DNA structure in a manner that it lengthen s the Other techniques can be applied to study the same phenomenon. One can determine whether the genome of an organism is circular or linear using this Time 5. DNA molecule is incubated with a nuclease. This can happen only when the length of DNA For this. with circular DNA molecules. the moment a double strand break results. proflavine binds to double-stranded DNA tightly. This is because the DNA will retain its configuration despite the single strand due to the rigidity of base stacking. multiple double strand breaks will eventually occur resulting in multiple linear fragments of smaller s izes. what should happen to the r of the solution? As long as the nuclease is causing single strand breaks. However. Thus. of DNA If a circular. increase in the axial ratio and the r will increase. the viscos ity will initially and then fall (Figure 5. When DNA is incuba ted with proflavine. .12). the viscosity will not increase at all if it is incub ated with a Initially. that Certain Dyes Intercalate in DNA The dye.141 not put in the mixture or if Mg2÷ is absent. Vscosi ty never Increes. With linear DNA molecules. noth ing much happen. nothing will happen and then the viscosity will start going down. will be rendered linear. From a plateau it starts decreasbg with time. This ca . (B) Linear DNA. Initially the viscosity increases. the sedimentation coefficient of the complex decreases. This will result in a decrease in viscosity. This will cause an. If the nuclease action is allowed to continue. When DNA is incubated with the viscosity of the complex increases. This proves that DNA polymerization all the components in the reaction mixture described above.12 (A) Circular DNA. viscosity should increase. the blood. Generally . It may also happen if the axial ratio o f the complex increases. one learns that the dye becomes immobilized a nd seems to be in the same place as the bases are. SIGNIFICANCE OF VISCOSITY IN BIOLOGICAL SYSTEMS The importance of viscosity in human health and disease stems from the fact that the vital fluid circulating through each and every part oF the human system. This is not observed. Employing fluorescec polarization. it seems that the second possibility is correct. This indicates that the dye gets in between the bases and pushes them apart thereby lexgthening the DNA chain. Th us. and this is observed. DNA length indeed increases when it is treated with this dye.n happen if DNA becomes depolymerlzed due to proflavine. With the second possibility. a decrease in viscosity must take place. is highly viscous and is affected profoundly by even a small change in the viscosity of the medium . Thus. With the first possibility. e. viscosity of blood decreases with increasing shearing stress. y = rate of shear and = shearing stress. Figures in rectangles indicate the diameter of t he tubes chosen for the experiment Blood.1161 / 1494Fm .. Temperature is regulated at 25°C. This anomalous beh aviour of blood is attributed to the erythrocytes as they are the predominant particles of blood .urn i1111 m115 Prn 0 " i 2O 40 60 8O Haematocrit (%) Figure 1 Plot of apparent viscosity of erythrocytes in physiological saline agai nst hematocrit. behaves as a New tonian fluid in blood vessels with internal diameter 1 nm and above. .3 Nature of Blood Flow through Different Vessels Newtonian fluids (viscosity independent of shearing stress) under conditions of laminar flow and constant temperature obey the relationship. But as the blood vessels get narrower. the percentage of the blood occupied by the erythrocytes. Box5.142 Biophysical Chemistry blood flow varies inversely with the viscosity of the blood.3). i.$ 945um 370. Viscosity depends o n the hematocrit value. one of th e major components of the blood. where q = viscosity. The apparent . The large vessels like the aorta and vein have a high hematocrit v alue whereas the capillaries and small veins have a very low value due to the difference in the n ature of flow of blood through the different vessels (see Box 5. though inhomogeneous due to the presence of blood cells.. In capil laries.B.s increases in the co re region and . Because of th is property and the fluid content of the cells. the erythrocytes assume shapes which facilitate their movement. The behaviour can be explained well if we go into the characteristics of the red blood cells. The red blood cells are biconcave having extremely flexible membranes.C. which have a diameter smaller than 15 m. Wit h increasing shearing stress.red cells align themselves obliquely along the tube axis. At smaller diameters.l) The figure indicates the change in apparent viscosity of blood with respect to h ematocrit values in vessels of varying internal diameters. the red cells undergo extreme deformation (they assume an "arrow head" shape). This pro cess is known as dal migration. AS a result. As the shearing stress in these vessels of narrow bore increases the.C.s have a high degree of fluidity.blood viscosity is indicated by the value of hematocrit which is about 45% for m an (see Fig. even upto 3 m. the R. flattening of th e curve is observed. the concentration of the R.IB. viscosity rises. These changes are of the reversible ty pe. Thus. diabetes mellitus. icterus and pneumonia. the jelly like gel state being converted to the fluid or sol state and vice versa. The cytoplasm in these organisms is sur rounded by a thin membrane. A thick rigid layer of plasmagel lies beneath th . The changes in Amoeba proteus have been well described by Mast (1932). leukocytes an d embryonic nerve cells has very close connection with viscosity changes. that the blood of patients suffering from congestive heart failure was more viscid than normal.The layers in the peripheral region now have a lower viscosity and a high shear rate This lowered viscosity facilitates the m ovement of the central column of blood (predominantly R. Blood viscosity is increased in several diseased states such as cyanosis. the r ed blood cells are very rigid and because of this abnormality. the average time spent by an erythrocyte in a narrow vessel like a capillary is shorter than its counterpart plasma.C. In seve re polycythemia.B. Variation in the viscosity of blood influences the working of the heart. polycythaemia. Apart from the above parameter. In severe anaemia. It was o bserved by Mackson in 1936.) with an increased velocity. Amoeboid movement which is a characteristic of all naked protozoa. The hematocrit value is therefore smaller in an acti ve capillary than the hematocrit of blood at rest. During t hese movements striking changes in viscosity take place. the volume element. the plasmalemma. The anaemic person presents a totally different but interesting picture. the blbod pressure and the hysteretic (elongatio n and shortening) behaviour of the smooth muscles forming the vessel wall are important determinan ts of the flow of blood through a blood vessel. multiple myeloma. mainly the immun oglobulins and other plasma proteins also play a significant role in altering the viscosity of blood leading to many pathological states. Besides the hematocr/t. peripheral resistance is decreased but the cardiac output increases resulting in overwork of the heart. the other constituents of blood plasma. the increase in viscosity of the blood increases the work of the heart.Vscositj 143 blood gets thinned out near the walls of the vessel. In diseases such as hereditary spherocytosis. m. 1953. the WBC. Tanford. New York. The plasmalemma however.e membrane.H. Protein Structure. R.. The plasmagel surrounds a fairly fluid plasmasol. Haschemeyer. The Viscosity of Macromolecules in Relation to Macromolecule s. Physical Chemistry of Macromolecules. . Proteins : A Guide to Study b y Physical and Chemical Methods. New York.. Shape and Hydration of Protein Molecules in The Proteins.T. Yang.E. H. Neurath and K. 1961. 5. 323 (1961).. Edsall. New York. 16. Protein Chem. J. New York. remains attached to the plasmagel and the su bstratum. The newly formed plasmasol at the retreating end now advances forwards and is transformed into the plasmagel at the front.. As locomotion begins the plasmagel at the retreating end is converted into the plasmasol. The forward flow of the plasmasol is facilitate d by the alternate contraction and relaxation of the plasmagel at the retreating and advancing ends respectively. Scheraga. 2. J.acrophage. connective tissue cells and malignant sarcoma cells during cell loc omotion and cell division. with a thin fluid hyaline layer sandwitched in between. 3. Academic. Wiley. Similar gelation changes are observed in many other cells of physiological origin i. Adv. Locomotion is thus affected by the reversible gel-sol transformations as explain ed above. Owing to these differences in the elastic strength of the two regions the cell i s propelled ahead. The Size. C.A. John Wiley. and Haschemeyer. 1973. eds.V. Academic. Vol. Suggestions For Further Study 1. 4. A. ] (H.T.e.. 196 I. Bkiley..). You are given an unknown proteip. the viscosity decreased. Suppose hat you have incubated a double stranded linear DNA with a nuclease manipulating the conditions so that one single stranded break results every 10 m inutes. No definite conclusions can be derived from this o bservation. how many possibilities can you imagine which can give rise to reduction in vlscosi! in such a situation? 6. Using viscosity measu rements how can you prove which is which? 3. . A DNA solution has inadvertently been mixed with another unknown chemical and a-reduction/ in viscosity can be observed. What possibilities will you consider about this protein based on thk¢ observation? 4. The optical density increases. or the temperature. is added to the solution and then the solution is heated. When one heats DNA. However. ionic strength.144 Biophysical Chemistry Problems I. What does this tell you about the structure of the polypeptide? 2. On an average. it takes about I 0 single stranded breaks before a double stranded b reak results. what should happen? If the salt concentration is increased. One can manipulate the activity of enzymes by manipulating the pH. If poly 7-benzyl-L-glutamate is denatured with 6 M guanidinium chloride the viscosit} decreases. What shou ld happen to viscosity under such a situation? When a salt. After what time do you think the viscosity of the solution undergo a significant reduction? 5. You are given a plasmid and a linear DNA fragment. it 'melts'. say NaCl.When you denatured it with 6 M guanidin ium chloride. what shou ld happen? 7. If a polypeptide is incubated with a protease. what should happen to the viscosity of the solution? . liquid. is not only s hapeless has lost its volume definition too. the addit ion of and translational energy makes them more mobile. As a result. and gas. Interfaces may be of various types. two gases do not form an interfac e of their high diffusibility. A n assembly of can exist in any of these states provided temperature and pressure are favourabl e to equilibrium which is established between the kinetic energy of the molecules and the forces between them determines the state of the matter. Further.6 SURFACE TENSION Dry sand. solid . This anomalous behaviour of the sand particles nearia surface and in the of the liquid is related to the phenomenon of surface tension. A gas.. A liquid has molecules arranged at an average constant distance but thei r do not remain flied due to 'not so strong' attractive forces. or between a liquid a gas is more commonly termed as a surface. we shall discuss some properties of matter and matter appears on a molecular scale. on the macroscopic the liquid is shapeless but has a definite volume. The water surface beh aves as flit were covered by a fine elastic skin. Before we proceed to what this phenomenon means. when sprinkled gently upon water readily floats. Also. or may not be chemically identical with each other. An interface made up of two phases o f the compound is highly dynamic in the sense that molecules are continuously leaving one entering into another and returning to the original phase. A solid has molecules co mpactly in a regular pattern. Matter may exist in three different states in nature . each state is called a phase. " In a system where matter is present in more than one state. however. All kinetic energy possessed by the molecules is confined to energy. When one moves from t he . The region of contact of two is referred to as an interface. An interface between a solid and a gas. viz.solid. two luids. if introduced below the surface r sink. It is because the molecules in the assembly are only attracted to each other and the translational energy of the particles has gained an upper over the attractive forces. The same sand particles. gas-liquid or gas-solid. Two solids cannot ordinarily come into close con tact trapping any gas or liquid between them. liquid. across the interface to the interior of the adjacent phase. and free forces of attractio n are not .. refractive index. at the int erface. viscosity. the molecules attract each other with a cohesi ve : which is uniform in all directions because they are surrounded by other molecu les of the kind. it is observed that in the body or phase. On examining the arrangement of molecules in a phase. absorption behaviour etc. heat capacity. take a liquid for example. one experiences an m many measurable properties such as density. the properties beg in to in accordance with the physical state and chemical nature of the material making up body of the individual phase. dielectr ic electrical conductivity. after moving a considerable distance away from the interface. They are thus free to move in all directions. The relationship between surfa . Both surface tension and surface eli iergy have the same dimension in the CGS system of grams per second2. the molecules are only partially surrounded by other molecules. liquid with surface tension of 60 dynes/cm possesses a free su rface energy value of 60 ergs/cm2. Numerically they have ide ntical values. The units are dynes/cm.. compression. Drops or bubbles canj molecules of a liquid so as to resume a variety of shapes under the Influenc result in surface tension of gravity. The special strain that the surface layer experiences as a consequence of the un balanced forces is called surface tension. they resume their spherical shape. and as a consequence they experience an inward pull (see Figure 6. for e.energy. It is much higher than the potential energy carried by the molecules in the body of the liquid.g. The result of this inward attraction is that the number of molecules at the surface tends to be reduced to a minimum and the surface contracts until its area is the smallest possible for a given volume of liquid. By definition it is the force in dynes acting along the surface at right angles [90°) to any line one cm in length. At the surface. The work required to increase the surface area by I square cm i n called the free surface energy. Therefore.on the other hand. I Cohesive forces acting upon falling water or oil drops. This behaviour of the surface is responsible for the nearly spherical shape of Figure 6. molecules have to be forced out from the interior to the top and this requires . The surfa ce possesses a considerable amount of potential energy. It !s because a sp here is a body having the least area for a given volume. The surface is eager to accommodate certain types of molecules (surface active) always in order to decrease its free surface energy. they are som etimes used synonymously for convenience during calculations. though surface tension is an intrinsic property of liquids and surface energy a fundamental property of the surface of the liquid. If the surface has to be extended. and stretching but a soon as they are released.1).146 Biophysical Chemistry exhibited. Work has to be done to overcome the attractive forces between the molec ules and create the new surface. It is expressed in ergs/cm. . the work done in increasin g the surface area by 1 sq cm will be ¥ ergs.. ff the surface tension of liquid is y dynes/cm.(I) dT d¥ where is the rate of change of surface tension with a change of temperature. surface area and the Surface free energy can be summarized as Surface Tension x Total Surface Area = Surface Free Energy Thus.. a heat term has to be added to the surface free energy. and is the total energy at the surface. however. and the surface free energy will be y ergs /cm2. As heat must be supplied to the expanding surface to keep the temp erature in the surface constant. .ce tension. The differential term is always negative as surface tension always decreases with an increase in temperature. may not be equal to the surface free energy alone. The total surface energy per unit area. The to tal energy at the surface per sq cm then will be equal to dy p=y-T -. higher is the surface tension. When two immiscible liquids come in contact wit each other.. For this of solid-liquid interface.2 = ].. it either spreads out into a thin fdm or a drop on the surface depending upon its affinity towards the solid surface. The molecules at the interface of two immiscible liquids are more or less satisf ied and to this decreased number of unbalanced forces. A angle (e) is the angle between a solid surface and a liquid meniscus (also see B ox 6. and Y2' = surface tension of liquid 2. interracil tension can be expressed in terms of conta ct angle.Box 6. /. This is because the dividing surface or interface contracts spontaneou sly in response to the attractive forces of the molecules in the interior of each liquid respect ively.1).2' .interracil tension between liquids 1 and 2. this interracil tension is not an additive property of the surface tension of the two different liquids. an interracil tensio n develops. It depends on the nat ure of the liquid. It is for the same reason that the potential energy at the surface of the molecules of each li quid is far below than that seen for molecules in the body of the two liquids individually. When a drop of liquid is placed gently on a slide. Greater the magnitude of the attractive forces operating between the mol ecules of a liquid.1 Surface Tension 147 The surface tension of a given liquid is a constant value. However. interracil tension is lowered. .. Useful information regarding the wetting quality of a surface can be obtained by . The relation between the interracil tension and the surface tension of the two i mmiscible liquids is given by Antonofi's rule which stands as ]. {2} where y. magnitude of this angle is influenced by the forces acting along the surface of the Solid.2 -. Instead it is seen that the value is often slightly lower than the larger of the two surface tensions. = surface tension of liquid 1. comprising of the monocytes and the macrophages. . For values above 90°. (see Box 6.1) Wetting of Solids and Contact Angle It haslong been surmised that interracil energies play an important role in phag ocytosis (engulfing and killing) of bacteria and other solid bodies by mammalian leukocytes. For phagocytos is to occur. It is one of the oldest techniques devised byYoung in 1805 for studying the natu re of solid surfaces. as the second line of defense. Naturally. as shown in the figure below. The solid-liquid interracil tensions of solids have been studied using contact a ngle measurements. For contact angle values upto 90° the extent of wetting is good but no t perfect.determining contact angle and there lies its importance. wetting is poor or non-existent. the l iquid wets the completely. If the contact angle is zero. A contact angle e is the angle between a solid surface and a liquid meniscus. the bacterium and the phagocyte have to be brought into sufficient contact with each other. Thus 'a knowledge about the interracil energies of the b acter um and the phagocyte can allow us to predict the extent of phagocytosis. It migh t be mentioned here that the mammalian phagocytic defense consists of the circulating polymorph onuclear leukocytes (PMNL) as the first line of defense and the reticuloendothelial syste m. the surface energy of the bacter um and the phagocyte are intimately related to the degree of phagocytosis. p![os p!nb!'I . .0° approx.g.6° Mycobacterium butyricum 70.3° Klebsiella pneumoniae and 17. (MV)2/a is proportio nal to the molecular surface. i carbon tetrachloride.0° Streptococcus pyogenes 21 3° Sa/monella typhimurium 20. e. surface tensio n has a temperature coefficient. i. alcohols.. at an y temperature t. benzene.. etc. K is a constant called the temperature coeffici ent of molar surface energy having a value of 2.0° Streptococcus pneumon. iae 17. The relationship between surface tension and temperatur e was first provided by Baron R. The relationship may be writt en as 7(MV)2/3 = K (to . For another batch of liquids like water. (3) where te represents the critical temperature of the liquid.149 Surface Tension Haemophilus influenzae (rough) 18. von Eotvos in 1886.e.0° Human Neutrophils 18. ethyl ether. .12 for a number of "normal" unassociated liqu ids. and 7 and V are surface tension and specific volume respectively.t) . expressed in degrees centigrade.. FACTORS AFFECTING SURFACE TENSION Temperature As temperature rises surface tension of a liquid decreases.2° Sa/rnonella arizonae 19. i..md carboxylic acids.. Van der Waals. 7 = to (] .t/to) . the temperature coefficient is not only less than 2.p. In a state the actual molecular weight of the liquid is not M but xM.6) .. The above particulars for explaining the nature of the temperature Cdefflcient were given by Ramsay : and Shields in 1893.. the molar :i surface energy could be expressed by a modified form as y(MV)2/a = K (t .) = K(t -t) = Kt (1..t/t)" .6) . Katayama proposed modified form of equation (3) which is 7----. (6) a universal constant and to depends on the critical constants of the liquid. the surface tension of a liquid at temperature 't' should to the critical temperature to by an equation of the form. The Ramsay and Shields equation then takes the form 7(M/P)21 = K(q . Later in 1916.e.t .D. 'n' has a close to 1.. They found that at temperatures not too near the critical point.t .12 but varies with This abnormal behaviour is attributed to the associated state of the liquids. (5) According to J.(7) p" are the densities of the liquid and its vapour respectively at the same tempe rature. M. the average number of single molecules combined to form an ag gregate. where "x" is the factor of association.. ...2. (4) is equal to molecular volume can also be written as M/p where p is equal to the of the liquid. Several liquids have shown a value of zero only at the critical tem perature and not earlier. but the effect is relatively small. is not univerl.. For such liquids. the surface tension of the liquid becomes zero when the temperature reaches a value which is lesser than the critical temperature to. (81 where C is a constant for each substance. It is obeyed with considerable accuracy by many organic compounds nearly upto their cr itical temperatures.Sudgen {1924) is essentially a molecular volume modified to eliminate some of the influence of the cohesive forces which are different for different liquids. The above predictio n. will have parachors of th e value P] = p] V]7/ and P2 -. Katayama's equation is found to be the most suita ble. we get MY i--= ME = P (constant) p-p' The constant 'P' also called the "parachor" by S. The above interesting relationship (8) between the surface tension and density o f a liquid is also called Macleod's equation (1923) and it holds over a wide range of tempe ratures. It follows that at critical temperature the surface tension of a normal liquid will become negative.. Two liquids of molecular weight M and M2 hav ing densities p and P2' and surface tensions 71 and 72 respectively. .92 V2 Y2]/ where V and V are the molar volumes of the two liquids. In equation (4). It is expressed as y'"/(p. by 6°C.p') = c . For associated liquids the value of C increases\ slightly as the temperature is raised.150 Biophysical Chemistry The above equation holds quite accurately for normal liquids at temperatures muc h nearer to the critical value than does the original form of Eotvos. If both the sides of equation (8} are multiplied by the molecular weight of the substance M. Parachor may be defined as the molar volume of a liquid at a temperature at which the surface tension is unity. however. ac cording to Ramsay and Shields. glycerine. the inward pull exerted by the molecules in the interior of the liquid upon those on the surface. amines.With an increase in temperature. proteins. The hydrocarbon residues in the fatty acids are not at ease in the interior of the solution and therefore if they are to be brought to the surface. Substances which decrease the surface t ension of a solvent are said to be cap///ary active. The polar part of the molecule makes it reasonably soluble. increase the surface tension of the solvent. Consider for example an aqueous solution of fatty acids. etc. Once they accumulate at the surface. The capillary . A majority of organic compounds are neither typically polar nor completely non-p olar. though slightly. and are therefore called cap///ary/nact/ve. is lessened.. and ketones belong to this class. alcohol s. kinetic energy of the molecules of the liquid I ncreases. esters. sugar.Surface Tension of a Solvent Lowering or raising the surface tension of a solvent on addition of a solute dep ends largely on the chemical structure of the solute. Hence a decrease in surface tension of a liquid is observed with risin g temperature. salts of o rganic acids. The fatty acids possess a hydrophflic head (polar) and a hydrocarbon chain (non-polar). Soaps. Effect of Solutes on the . This will weaken the cohesive forces acting between the different molecules. surface tension of the solvent will be lowe red. little work will be required. ethers. As a result. organic acids. Other like inorganic salts. bases of low molar mass. A similar movement towards the surface is seen when non-polar olutes are dissolved in water. Saponin. by virtue of ion-dipole attractions. The graph shows three types of Type I curve corresponds to surface inactive substances. The a lbdmin in it accumulates at the surface of each bubble as a result of surface concentration md coagulates there. nd bile salts have remarkable surface tension lowering ability. However. Positive adsorption can be visualized in the form of a surface crust when beaten white of egg is lef t standing. inorganic electrolytes in general. raise the surface tension of water and are. a plant glucoside. if the surface has to be extended. In as much as surface tension is primarily dependent on mutual attraction of molecules. As a result a rigid film is formed at the surface. salts which are ionic and therefore polar in nature. the curve is remarkable in the sen se that the in surface tension occurs only upto a particular concentration. Beyond this.I Surface Tension 151 activity of these amphipathic molecules thus depends largely on the predominance of the non of the molecule.2. The increase in surface tension concentration of these substance is infinite. In such a situation. any in concentration will not alter the surface tension of the liquid. A very dilute so lution of saponin In water exhibits a surface tension of only 20 dynes per cm as compared to 73 dy nes per cm of pure water. These solutes concentrate preferentiallyat the su rface and lower the surface tension of the solvent. . Type II curve the changes in surface tension of a liquid on addition of non-electrolytes or we ak The graph depicts a gradual and regular decrease in surface tension of solvent olute addition. addonal work be done to overcome the electrostatic forces. They are then said to be positively adsorbed . tend to pull the water mol ecules into the of the solution. On the other hand. accordingly. negatively adsorbed at a water surface. An opposite effect occurs on addit ion surface active substance (type Ill). Polar substances are therefore. ca pillary above observations are summarized in Figure 6. Figure 6.}. II Non-electrolytes and weak electrolytes.2 Effect of dlssolved substances on the swface tension of the solvent. I Surface inactive substances [strong electrolytes {ionic salts} alkali]. etc. Ill Surface active substances {soaps.Concentration Figure 6. proteins.3 shows surface tension of aqueous solutions of amino acids varies with concentration of the . The behaviour of amino acids also differ according to their polar nature. I 152 Biophysical Chemistry Solutes which show a decrease of surface tension with increasing concentration a re said to exhibit positive surface activity. The solutes of the former type arc Concentration Figure 6.amino butyric acid Ill a-alanine VII y . Box 6.2 .. the respiratory organs agraduatedseries oftubes that medium cimulates through these airways usually occurs by means of diffusi and relaxation (inspiration) of the alveoli. while those that raise it are said to have a negative surface activity.. I Glyclne V Lysine II -alanine VI y .amino caproic acid IV -aminobutyric acid Solutes which show a decrease of surface tension with increasing concentration a re said to exhibit positive surface activity. The role o f surfactants has been extensively studied in man and it is seen that lung function is intimately linked with the amount of surfactant present in the alveoli (see Box 6.2).3 Surface tensions of amino acid solutions at 25°C.. while those that raise it are said to have a negative surface activity. The solutes of the former type are also called surfactants. Role of Pulmonary Surfactant: Stablllty of Alveoll Lungs. The alveoli are during expiratioD the expiratory muscles . Thisproblem iscircumvented by mixture oHipidsand proteins..the. the interfacewithin'.aqueous film or the.pressure suffiCletlyto. < . Because tl to theradius accord=n. -- end expiration would lead to as the smller alveoli wou!d empty into the larger alveoli.the surface tension generated bythethin . andso on.to katlace's lawwhiCh is given as . eollapsethe alveoli. . A consequ ence of this is the respiratory Ji distresSsyndrome (RDS)wh ch isa eading cause of morb d tyjn these infant s. leading to their increased acumu ation!n th .rmaintaining t. Pulmonary surfactant is acomplex mixtureof about 90% lipid 10% protein and small pementage lmitoylphosphatidylcho ine generated by the aqueous . the normal pattern of ung development s fii: = interrupted frequentiyresulting in suffactant insufficiency.153 Surface Tension of thelung at the end of each expiration. An established [. .r.: ..This =treatmitis reported to have sho wn an alteration n the levels of surfactnt protein. but investigations regarding [ :n=sare inco studies on surfactant proteins suggesttht these . a direct relationship has been reported between the amou nt of surface active mater a 11 : and the pulmonarysurface area. like collapsed -balloons would require-greater pressure to inflate them than partially collapsed alveo i and th is would hamper smooth:gaseous exchange. treatment of this Complication is introduct on oflhigh concentrations of oxygen through nspiration " accompanied by ventilatory support.the . prgteins may play a prominent role in tPie organization of thelipids int he airway I' In man.yelin is. In premature infants..i. The collapsed alveoli. .elngs: However. It is this property that is respon sible to some extent in forming the limiting membranes (see Box 6.: .-i regarding the regulation and expiession of the genes itiv01ved in surfactan t protein synthesisand enzymes involved in surfactant phospho!ipid synthesis bef ore th s approach can be applied The tendency of molecules to either concentrate at the surface or remain in the interior assumes great significance in the human body.the iil : .3).. An alternate approach for i.. namelyphospholipid and thesurfactant"prote us.= treatment of the diseae lnvolvetracheal administraton of multipledoseof mixture of... current . Biomembranes show proteins . surfactant materials. " resolution of acuIeRDS inv es plarmacoiogical internt on but mCich rema u s to I: elucidated . 1972 (see Figure 2) .. electro n microscopy and others have revealed the dynamic nature of thecell membranes which have been summarized in the Singer and Nicolson's fluid mosaic model proposed in.for cell membrane . nuclear magnetic r esonance (NMR) " and electron spin resonance (ESR) spectroscopy.biophysical techniques involving X-ray diffraction. '] ' Protein s Figure 2 The Singer and Nicolson model." backbone - : Llpold film { chainSpolar Hydrocarbon ]head ---.-fluorescence microscopy. Exterior Protein film Hyd}ocarbon chains Polar head Lipold film Protein film Interior Figure 1 TheDanielt and Davson model for cell membrane Untilthe late 1960's bilayer models were often pemeived to be static structures but studies with a series of.154 Bio1hysicca Chemistry proteins adhering to lipid-aqueous interfaces because very small amount of prote ins were effective in reducing the surface tension of a model ]ilid-water system considerably (see Figure 1). Carbohydrate chains Proteins attached to polypeptide \i 3-'. The opposite is observed for a concave liquid surface. When liquid from a plane surface forms a drop. st amongst the three surfaces. The drops also have a higher vapour pressure than earlier (plane surface). Whenwa ter is in a pipette.4}. and in the form of drops it has a convex surface (see Figure 6. A minimum in the surface tension Is observed in the isoelectric zone. Surface Tension and Vapour Pressure The surface of a liquid assumes different shapes in different situations. it has a concave surface. . it has a p lane surface.Hydrogen-Ion Concentration Surface tension of protein solutions changes with hydrogen ion concentration. There. The molecules of the convex surface are attracted by other liquid molecules to a less extent now than they were in a plane surface. This increase requires more surface energy. t he surface liquid molecules are attracted more than they are in a plane surface. These observations are compiled in an e quation given by Kelvin. When on a glass slide as a film. there is an increase in surface area. The vapour pressu re here is also the lowe. Among the static methods. The Kelvin equation indicates that liquid drops have a considerably higher vapov . and r is the radius of the liquid drop or the cap illary (if it is oncave surface. y is the surface tension in dynes cm-.. static methods are based on the assumption that the liquid has attained sur face equilibrium. MEASUREMENT OF SURFACE TENSION tension of liquids can be measured by either of the tv. the most commonly used ones are.. There are other methods too which fall between the static and the methods. p is density of the liquid. r is negative).Tension 155 P 2M7 In Po RTpr . The dynamic methods measure the tension of a liquid before the surfac e has had time to form. (10) Po are the vapour pressures of the plane surface of the liquid and convex surfac e drop) respectively. R is the gas c. uids and solutions of crystalloids the process of attainment. (AJ Concave surface when in a pipette.4 Surface of llquid JIlms. of equilibrium is very the static methods are best suitable. But for colloidal solutions a considerable time is reach the equflibrium state and therefore the dynamic methods of measuring surfa ce preferred. () lgure 6. methods: static and dyna mic.r pressure () @ @ O0 . M is the molecular weight. {C) Convex surface when as a drop.(/) the capil lary . (B) Plane surface when as aJIlm on a glass slide.onstant expressed in ergs per degree per m ole (8.31 x T is the absolute temperature. (it/) the Wilhelmy balance method.(//) the du Nouy ring method. and (iv) the dr op- . e. This' force-area relatioriship can be recorded as a curve on a kymograph. A lens system is placed in such a way that it directs a beam of light from a light source on this mirror. 1 967.. surface tension as a function of area. New York. Reflected beam of light now illuminates a tw in phototube.J. London.ansformed into an elec trical message which is captured in a potentiometer recorder. In 1963. Any change in the film pressure displaces the light beam and this serves as a si gnal to the phototubes to bring about the firing of either of the two thyratron tubes which in turn activate a motor. the angular dplacement taking place is tr . and Vold. Study of hysteresis loops.J Introduction to Colloid and Surface Chemistry.Shaw.In another instrument described by Anderson and Evett a mirror is attached to the horizonta l float of a horizontal idm balance. i. Colloid Chemistry. responsible for the pressure-vo lume hysteresis following contraction and relaxation of the whole lung. Physical Chemistry of Surfaces. 2.W. Vold.D. Reinhold Publishing Corpor ation. 1966. John Wiley & Sons. . . M. In doing this. New York. are of physiological importa nce. 3. 1964. R. The se automatic devices record the pressure exerted by a fflm as a function of the area occupied .. Adamson. Mendenhall and Mendenhall described an instrument which can carry out t he compression and expansion cycles of surface iflms automatically upto a ratio of 70:1. The motor works to restore the float to its original position by means of a torsion wire. Surface pressure may be changed by altering the area occupied by a film either by compressing or expanding the iflrn.2). It has been observed that a surfactant found in the alveoli. This phenomenon of alternate compression and expansion is called hysteresis. Suggestions For Further Reading 1. D. A. Butterworth.156 Biophysical Chemistry Automated Recording Devices In the middle of the 20th century many automatic recording instruments making us e of either the Wilhelmy vertical pull method or the horizontal one were devised... exhibits hysteretic beha viour and thus prevents the lung from collapsing after a contraction wave (see Box 6. .E. andWhitsett.. Weaver.Associated Proteins. Biochemical Journal. 249-264 (1991).A. J. Function and Regulation of Expression of Pulmonary Surfactant -.4. 273. T. This phenomenon of concentration of molecules or long on the surfaces o f all is called adsorption.7 ADSORPTION In the preceding chapter on surface tension we discussed the nature of cohesive forces between the molecules of liquids and solids.2). surfaces of liquids tend to attract and retain on them gases or dissolved substances with which they contact.t Figure 7. In order to satisfy their residual forces. In the latter proc ess a ' retainedon the surface. I Adsorption : Concentration at tim surface Adsorption should be carefully distinguished from absorption. The substance which is thus adhered to the surface is said to be the phase or adsorbate while the substance to which it is attached is the adsorbent (see 7. However. at times. but penetrates to the interior to become distributed the phase (see Figure 7.Surface . " o //////////S//////////// AdsorUe.W. -. 1909) is implied in such cases. both adsorption and absorption ta ke side by side and it is difficult to distinguish between the two processes experi mentally. and thus bonding forces at the urfaces are not completely satisfied.McBain. general term sorption (J. We observed that surface atoms cann ot in the three dimensions as the atoms in the interior do.1). . a constantly changing electromagnetic field is set up outs ide the substance. Instead.r instantaneous dipoles. These forces arise from attraction be tween temporary . their motions are no longer indep endent. 158 Biohysico Chem/stry KINDS OF ADSORPTION INTERACTIONS The forces operating between the molecules of an adsorbate and the adsorbent are mostly short-range forces as suggested by I. The physical forces involved in the adsorption process are non-specific in natur e like the dispersion forces or van der Waals forces. the orientation forces and the Induction forces. when a strong chemical bond appears between a molecule of the adsorbate and the surface of the adsorbent.3). These forces are called dlspersionforces because fluctuating dipoles cause the phenome non of dispersion 11) 12) 13) iVgure 7. When two dipoles approach each other. At any gtven instant it may influence upon the electrons in neighbouring matter in a way that the electron cloud may be more concentrated at one side of the atom producing fl uctuating dipoles.3 Model of dlspersion forces (I) Even distribution of electron density of an isolated atom. In such a case. the phenomenon is called chemical ad sorption or chemisorption. The origin of an important part of the van der Waals f orce can be elaborated thus: owing to the continuing motion of all the electrons in their or bits within the atoms of a substance. two dipoles oriented in the same direction produce an attractive force (see Figu re 7. These forces may be non-specific like the dispersion forces.of light. Sometimes ch emical bonds like ionic and covalent bonds which are specific in nature may also be involved in th e adsorption process. . Langmuir. (2) Instantaneous dlpole formatlon due to uneven distrtbutlon of electrona. Polar molecules tend to orient.4). but somewhat stronger than the dispersion for ces between non-polar molecules. Polar molecules (these have a net separation of positive and negative charge in individual molecules) have weak electrostatic forces between molecules resulting from attra ction between opposite ends of individual molecules. These orientation forces are much weaker than the electrostatic forces of attraction between long. For example. . themselve s with their positive poles toward the negatively charged surface or negative poles toward th e positively charged surface (see Figure 7. These hyflroxyl long holds on to the surface conferring upon it a ne gative charge.(3) Attraction of two instantaneous dipoles. if placed in an aqenvironment is negatively charged because certain valence of the carbon ato ms (secondary valence) at the surface which are incompletely satisfied attract hydr oxyl long from the medium. Other physical forces of attraction like the orientation forces appear upon adso rption of polar molecules carrying constant electrical charges. The electrical charges app ear on surfaces either as a result of electrolytic dissociation or preferential attraction of ch arged long from the sunndings incompletely satisfied atoms of the surface. charcoal. adsorbate may not contn dipoles to . It may so happen that dipole moments appe adrbent suace due to inducHon by the dipoles berg adsorbed. o o o o o I Adsorbate molecules 0-. AdsoHon d desoHon conue till is reached.159 ( Polar molecule circles represent adsorbent molecules Surface . centres (see Figure 7. but e ele cc chge on the suace of the adsorbent may duce dipoles in the molecules ereby attracting them. I 0 0 0 0 I..=: Surface ..40ntatn of molecs ducon forces so play pot role e adsofion process. the above e opposed by forces of repulsion. e cled inductive effects. e elecosc which elecons ier at tracted to or repelled fro one atom (or groups of atoms) to oer.5). e molecules of e adsorbate approach those of the adsorbent suace.. A molecule being adsorbed is not associated a sine cene. Iositively negatively : chged chged " (A) e 7.. Dotted lines represent residual forces of attraction of surface molecules ..-----.. if t he adsorbent of donor-acceptor interactions with the adsorbate. between two water mo lecules... H Figure 7. . Hydrogen bonds are much weaker than normal bonds but are stronger than thee/dipole-dipole interactions between polar molecu les. bonding occurs beositively charged hydrogen atom in one molecule and a highly electronegtive atom in a second m61ecule (see Figure 7.Figure 7..5 Adsorption of adsorbate molecules at multiple sites of the adsorbent In addition to the non-specific interactions discussed earlier.0 --H... unstable complexes are usuall y H -. 6)... molecular comple xes with bonds are often formed on adsor)ents.6 Hydrogen bonding represented by the doffed line. Further. physical adsorption proceeds best at low temperature and decreases with increasing temperature ([ Chatelier's principle).. However. ADSORPTION CHARACTERISTICS As adsorption phenomenon involves concentration of a substance on the surface. The larger the surface of the adsorbing agent. Therefore. Therefore. silica gel etc. 1 Kl mole-). .50-100 kcal mole-). t he extent of adsorption depends on the surface area. sometimes the association of the adsorbate and adsorbent may result in a strong chemical bond. In contrast to physical adsorption. fuller's earth. The latter occurs due to the increased kinetic energy of the adsorbed molecules which causes them to escape more rapidl y from the adsorbing surface. Physical adsorption can also be easily reversed by lowering t he pressure. the process is reversed. provide a large surface area and are best solid adsorbents. The chemical bonds formed between the adsorbate and the adsorbent cannot be broken easily and therefore chemisorption is seldom reversible. matter in finely divided or colloidal state exhibits great powers of adsorption. The heat o f adsorption decreases as the amount of substance adsorbed increases. Only a fter heating to high temperatures. Some systems may show physisorption at low temperatures and chemisorp tion at some higher temperature. Finely divided metals like nickel and platinum and porous substances like charcoal. These forces are characterized by low heats of ad sorption (approx. the greater is the adsorption. it may happen that the subs tance desorbed at higher temperature is not the same as the one originally adsorbed. apparently due to progr essively decreasing attractive forces as multilayers of molecules are built up on the sur face of the adsorbent.160 Biophysical Chemistry Finally. chemisorption is associated with high heats of adsorption (approx. Physical adsorption is due to the operation of relatively weak electrostatic for ces of attraction like the van der Waals forces. In such a case a new surface chemical compound is formed which ma y further take part in the adsorption phenomena. This orderly arrangement of molecules in surface films or at interfaces is called molecular o rientation. MOLECULAR ORIENTATION The molecules in the bulk of a liquid are randomly distributed relative to each other whereas they are arranged in more or less orderly fashion at surfaces or interfa ces. chcoal gets)activated when heated in streaming steam and then in a clos ed chamber at about 800/ .e.g.Adsorption proceeds best from dilute solutions.. The adsorbing powers of many substances can be increased by temperature treatmen t. i. it is more nearly complete when the ratio of adsorbent to the adsorbate is high. In the biological systems the patterned and orderly arrangement of the molecules of the cell membrane affords useful electrical properties. They are oriented a nd arranged in a definite manner relative to the adsorbing surface as well as to th emselves. The molecules adsorbed on a surface show a distinct pattern. As for e. This heightening in adsorbing powers is attributed to an alteration in the nature of the adsorbing s urfaces.. The surface charge of the adsorbent is an important criterion in adsorption. For example. A n egatively charged surface will adhere upon it more of a positively charged adsorbate than it will do of a negatively charged one. The . Heating in specified conditions may result in activation of the adsorbent. We shall discuss this aspect of adsorption in the subsequent text. cellulose strips will adsorb more of the dy e methylene blue (positively charged) than of congo red (negatively charged). acid molecules are attracted towards water and therefore they orient towards water through hydrogen bonds. -NO2. Because of the free pairs of electrons the s are surrounded by rather strong forces of attraction. if one of the earlier mentioned polar groups are introduced into a hydrocarbon m olecule. On the other hand. with their polar heads toward water and the hydrocarbon tails towar d (see Figure 7. -SO3H. When such a substituted molecule is added to water it surface in a characteristic manner. Molecular orientation results due to the differences between the forces of attra ction of the semipolar. On the other hand. -CHO. -NH2. Consider. This is so they contain atoms that have nearly equal attraction for the electrons and thus there is any difference in the electronegativities. etc. they do orient themselves at the wate r- .COOH) and propionic ac id are freely soluble in water. The acid molecules being freely soluble in benzene wh ile less : in water are retained in the benzene phase.. and non-polar groups in a substance for another substance which may b e the or ariother phase.7). But at the interface between the t wo liquids of caproic acid molecules takes place. An emulsion soon with benzene droplets surrounded by water. -CN. When a molecule containing one of the polar groups and a non-polar hydrocarbon radical comes in contact with a it will tend to orient itself in a definite fashion. The acid molecules thUS concentrate at the-interface of the two liqu ids. has greater affinity for the organic solvent benzene an d is towards it. The atoms that possess free pairs of ele ctrons a tendency to form co-ordinate linkages. the C5HI group acid. Nevertheless. for example. The polar groups like -OH. -COOH. contain atoms which do. a solution acid [CHc(CH)4-COOH] in benzene which is added to water.. Figure 7.7 Orientation of caproic acid molecules across a benzene-water interfac e Acids with short hydrocarbon chains like acetic acid (CH3. a non-polar group. a net of positive and negative chmges occurs. Hydrocarbons are characteristically insoluble in water but soluble in organic so lvents. The . Adam and their co-workers. and benzene. The benzene droplets thus form one ph ase water forms the other.161 of molecular orientation was In-st introduced by Hardy and further developed by Harkins. nonlike hydrocarbon radicals have weak forces of attraction around them. As a r esult. The arrangement is interesting.not have identical eectronegativities. with their polar groups adhering into the water while the hydrocarbon radicals a re . 8 Orientation of propionic acid molecules across a water-air-surface Membranes The living membranes have various kinds of molecules embedded in them. .Surface 162 Biophysical Chemistry directed above the surface towards the air vertically in a more or less parallel fashion (see Figure 7. There also exist macromolecules like proteins a nd compound which are polar at some of their atomic groupings but are non-pglar at o thers. On the hydrophilic heads are adso rbed the protein molecules. The arrangement of macromolecules relative to each other in the protoplasmic mem branes is worth mentioning. According to the Singer and Nicolson model proposed in 1972 . as influenced by environmental and intracellular conditions. Also. The protein molecules are so arranged because they are more hydro philic than the lipids and major part of the water of the membrane tends to be associated wi th the protein. Some are truly polar while some*fire non-polar. This lipids difference in size. while the similarl y shaped molecules of sphingomyetins and glycolipids tend to orient with their long axis perpendicu lar to the water surface. Solution of propionic acid CHCOOH in water Figure 7.8). For example. lecithins and cephalins have been seen to orient thems elves at a 0 water-lipid interface at an angle of 30 to the water surface. the lipid molecules are arranged in two layers with their hydrophllic heads to the exterio r while the hydrophobic hydrocarbon tails face each other. the molecular pattern due to orientation is constantly under chan ge. Protein molecules may also be an integral part of the bilayer (see Chapter 6 for diagrams). shape and arrangement of atoms in the different molecules in fluences their orientation. The protein molecules are capable of translation or rotation. Emulsions . the dr oplets come in contact with each other and merge to form larger particles in order that the tot al surface free energy may be reduced. The coalescence process is rapid and within a few minute s the droplets merge to revert to a two-phase system. one of the liquids is water an d the other liquid Immiscible with water can be designated as 'oil' for theoretical purposes. As soon as the mechanicai-ffispersive action ceases. This is an emulsion. The long hydrocarb on chains of the lipid molecules are bound together at many points along their long axis by h ydrophobic bonds. Accordin gly.rsion odroplets of one liquid in a nother may form. The membrane is dynamic and this poised arrangement of molecules may beco me disordered under certain conditions and a transition may occur from the gel-like ordered phase of the bilayer to the sol-like disordered phase. Usually. Any two immiscible liquids normally forti a two-phase system when mixed together. we come across . But by vigorous shaking a colloidal dispe. the liquid which has a hi gher tension will tend to draw itself into spheres and will be surrounded by the liqu id whose tension was more markedly lowered (see Figure 7. bile salts. betwee n lead to 'OfvV' emulsions. Milk is an excellent example o f an emulsion of butter fat in water stabilized by casein. Soap molecules Figure 7.e. By obeying the principle of minimal surfaces.Adsorption 163 emulsions belonging to either of the two classes water-in-oil type (W/O) or oilin-water type We have just mentioned that emulsions are usually unstable. The ir eiliciency lies in their preferential adsorption for one of the two interfaces. i . The ones which show a low HLB number. Most of the su rface-active (those possessing surface tension lowering ability) act as good emulsifiers. Surface-active substances can be put into two categories depend ing upon their number (hydrophile-lipophile balance number). a protein.9). The HLB number is calculated addit ively composition of their chemical structure. and saponins. A soa consists of a long hydrophobic hydrocarbon chain and a hydrophilic polar end (head).. the polar head b eing attracted to water will orient itself towards water and the non-polar tail will point the oil. i. We frequently come across m any other stabilizing agents such as gums. Such a dditional components are called emulsifiers or stabilizers. to six. i.9 Orientation of molecules in an oil-soap-wter system . oil-air or water-This is because the emulsifier is adsorbed on the drops d uring the formation of the emulsion reduces their degree of coalescence though it may later on go and lodge itself a t the oilwater interface. In a oil-soap-water system.. But there are agents which when added to such 'temporary' dispersions convert them into stable ones. lead to 'W/O' emulsions while those with a high HLB number. soaps..e. Ross and co-workers have shown that the HLB number to the spreading coefficient and hence in the case of O/W emulsions it is linear ly to the tendency of an oil drop to spread over the surface of an aqueous solution of the The most important aspect of the emulsifier action appears to be its orientation at the between the two liquids.e. On the other hand. it will b e an oill (Figure 7.10)./ Thus the emulsifier which is. Harkins pointed out that if the of the two parts of the stabilizing soap molecules is such that the polar end is chain. then a stable emulsion will be obtained in which the area of of the soap film is greater than that of the oil side. making it difficult for the surface of one droplet to the surface of another. In other words. added tends to collectt the interface between the phase (oil in this case) and the continuous-plse (water. in this case) to form a of coating on the surface of the droplet. thus preventing coalescence. if the polar group of the soap ha a smaller . but with the soaps of multivalent metals there wi .11). I0 An oil-in-water emulsion Soap molecules with a small polar head and a long hydrocarbon tail F/gure 7.WATER. in the case of univalent metal soap. According to this. but when calcium soaps were used. / 1 A water-in-oil emulsion It was observed that ffan oil was emulsified with water containing a sodium soap . Harkins forwarded the theory of oriented molecular wedge to explain this phenomenon. emulsions of the oil-in-water type resulted. Soap molecules with a large polar head and a smaller hydrocarbon tail Figure 7. each metal i on is attached to only one hydrocarbon chain. SOAP FILM SOAP FILM (1) Abundance ofsodlum soap in (2) (3) a off-soap-water system resulting in a oil-in-water emulsion 164 Biophysical Chemistry cross-section than the hydrocarbon portion.. the water-i n-oil type of emulsion was formed. then a water-in-oil emulsion will be stabilized {Figure 7. al. cal cium soaps are more soluble in oil than in water. Therefore. ther e are exceptions to this theory as can be pointed out by the emulsions stabilized by silver soaps . The hydrocarbon chains thus have a larger cross-section than the multivalent cation and this acc ounts for the stability ofwater-in-tl emulsions. Bancraft offered a different explanation for the formation of two types of emulsions stabilized by soaps carrying differe nt cations. silver soaps ought to stabilize off-in-water emulsions but these s oaps have been seen to actually stabilize the emulsions of the reverse type. sin soap to calcium soap producing no emulsion Abundance of calcium s o a p in a oil-soap-water system resulting in awater-in-oil emulsion . By this orientation wedge theory. while calcium soaps decrease the surf ace tension of oil and stabilize water-in-oil emulsions (see Figure 7. This theory has been tested experimentally an d proved to be true with various oleates and stearates by J. However. sodium soaps lower the surface ten sion of water sufficiently to enable it to emulsify fat.12).ll be several chains for each cation depending on the individual valencies of the metal long. Hildebrand et. whereas a sodium soap is more soluble in water than in oil. As per his suggestions.H. Critical rail. Various long have ant agonistic plysiologlca2 actlons upon p'otoplasm. any preponderance of sodium I soap yields emulsions of off-in-water type. calcium. Magnesium in high concentration in blood leads to p'ofound anaesthesia. etc. aluminium. If calcium is preponderant a bove this I ratio.. but toxicity is reduced considerably if the increased concentration is accompanied by a corresponding increase in the concentration of other long.g. magnesium to calcium and sodium to potassium. calcium is antagonistic to s odium. For example. like potassium I n thls case. Further work of Clowes on emulsions of both types gave interesting imdings. the emulsifying action of the combination of soaps will depend upon the ratio of metal long. an emulsion with oil as the continuous while phase results. For example. e.Adsorption 16 If in an off-soap-water system. iron. This inversion is due to a phenomenon known as /on antagonsrr Long frequently exhibit an antagonistic action. the proportion of calcium soap to sodium soap is varied. Sodium in hgh concentration s toxi c to the living cell. This critical ratio was Ca÷ : Na÷ = 1 : 4. but Intr oduction of . it was observed that an off-in-water type emulsion with a sodium or potassium so ap as emulsifier i may be converted into a water-in-oil system by the addition of salts ofbi-and te r-valent cations. i Clowes found out a critical ratio of calcium ion to sodium ion experimentally wh ich produced no emulsion. K. A monomolecular layer can be formed on the surface of water by dropping onto the clean surface a solution of the substance in some volatile solvent such as benzene or petroleum ether. Molecules continuously leaving and entering it. molecules in an oriented layer at an interface are in a dynamic conditio n. This-device is named after him as the Langmuir trough. or m0nolayer. water in contact with water vapour. Calcium long have a stimulating effect while potassium long have an inhibiting action on the heart. Teflon or Nylon)which is not wetted by the solutio/n. The trugh is coated with some material (Lucite. Sodium long increase the permeability of cell membranes to water soluble substan ces while i calcium long decrease permeability. Lastly. can exist only at interfaces. A c oating of Teflon is preferable as it is very hydrophobic and permits the study of flls under high .small amounts of calcium may result in prompt awakening and return to sensitivit y. the solvent evaporates and the subs tance is left on the urface of the liquid (water. in this case} as an insoluble unimolecular film. A much less distu rbed interface is that between a solid and liquid or between two liquids which are usually inso luble. Interfaces be tween two hases of the same compound (ice in water. An increase in temperature disrupts orient ation by increasing the kinetic motion of molecules. et c. The added solution spreads. Ir ving Langmuir. He evelo ped a device by which the two-dimensional pressure due to the spreading tendency of the hsoluble substance could be measured. These interactions of Na. Ca and Mg long in living organisms take an essential part in vital activities. most of the data available to date have been recorded either on unimolecular fil ms spread on liquid surfaces (liquid-air interfaces) or films transferred on to solid surface s. However. It con sists of a rectangular shallow trough. one centimeter to a few millimeters deep. The concentrations of thes e long and their relative proportions in tissues and fluids are of such fundamental importance th at slight variations in their content or composition produce profound changes in physlolog lcal behaviour. the American physical chemist in 1916 introduced new techniques and made some va luable and ingenious theoretical and experimental contributions to this field. Monolayers A monomolecular layer.) are highly dynamic and are continuously changing in molecular dimensions. pressure without leaks developing. The trough is filled to the brim with the liquid upon which the films are going to be . Quarter troughs may be used for study of protein films and metallic troughs avoided since small amounts of metallic salts have a stron g influence on the properties of films. The surface of the solution is separ ated into two parts by means of a teflon-coated aluminium or mica float. The float also has two non-wettable ribbons T1 and T connected to the sides of the trough.14).14 Side-view of a Langmuir trough. A torsion wire W. W is the torsion wire. This oper ation is repeated with another barrier B from the other end of the trough.film trough A and B are movable barriers.13). A third barrie r C. \ j Once the surface of the liquid is cleaned. F. C is the compression bar. which is al so non-wettable (see Figure 7.13 Schematic view of a simple. a film can be formed on its surface i . It can be moved horizontally to decrease or increase the film covered ea {Figure 7. The surface of the liquid is cleaned by a movable barrier A. is connected to the float.loat.Liquid 166 Biophysical Chemtstnj formed. F/gure 7. The movable barrier can be moved n either direction (indicated by arrows ) to increase or decrease the . F is the. The barrier is left a few centimeters away from the end of the trough.film covered area. also called a "compression bar" is then placed in position. TI and T2 are barrier ribbons. Movable barrler Figure 7. Oleic acid. for exampl e. The material to be spread in the solid. which is only slightly soluble in water spreads spontaneously In water. is bro ught into contact with the surface and will then spontaneously form a film. like higher sa turated fatty acids have to be first dissolved in organic solvents and then a drop of the solu tion is placed on . Others.n different ways. dissolved. or liquid state. In this way a number of protein stacked one above another. is very volatile and lighter than wate r. For certain studies. . a (coated with calcium stearate/barium stearatel can be stml. It is then removed. Certain proteins can be spread the water surface from the solid state. The choice of solvent is important. washed with water. It is not always necessary for the slide to remain vertical during the transfer process. The spreading of proteins should be carefully attempted. One should cho ose a solvent which does not dissolve in water. In th is. A monolayer ofundenatured protein molecules can be found adher ing the slide.] l. The ellipsome ter is a designed by Rothen which is capable of measuring a film thickness within + 0. but. it is advantageous to transfer the films from the liquid {water) surfac e to and has enabled a variety of studies on properties of deposited polished clean glass slide is first conditioned with a layer of calcium or bariu m stearate. It is also possible to transfer a monolayer or a system of stacked monolayers fr om one another surface. The complete process can be followed iran ellipsometer. then dipped down vertically into a protein film-covered water surface. agai n layer will be deposited on the uptrip and so on. and dried. This reduces the surface tension of the solution considera bly it to spread rapidly on the water. especially the determination of film thick ness methods. of undenatured protein molecules can be deposited on slides by adsorption.Adsorption 167 water surface to form a film. it is preferable to spread them fro m a dilute (0. It measures the change that takes place in ellipticity of reflected light produced by a deposited film. A protein monolayer deposited on the slide. tk i trough containing the protein solution for a minute or so. This difficulty can be circumvented by adding a small amount of amy l the protein solution. The ellipticity and azimuthal position in space of the reflected beam is a very sensitive and (over a certain range} function of film thickness. The spreading of globulins poses problems.05% or less} with the help of micropipette.3/. as in their native state the molecules are water-soluble but on unfold ing they ' insoluble.surface. Because the test slide has a hydrophobic surface due to the a layer will be deposited during the first downtrip of the dipping process. Deviations from this law occur of both the distribution of the molecules and the orientation of the molecules i n the film.A monolayer of matter behaves thermodynamically in two dimensions very similar t o three dimensional matter.. the precise number of molecules on the surface. If the surface concentration is small enough. experiments on monomolecular layers can yield valuable result s for of molecular cross-sections and for the magnitude of molecular interactions.15}.ogl cules were pushed together in the stacking position {see Figure 7. he prepared a mono!ayer of stearic/ cid [CH3{CH2}6COOH] on water. All the th ree quantities. This data is enough to derive the value of the cross-sectio nal area of the molecules when held in the upright position. /. The above dimension to the use of monolayers was provided by the pioneering experiments of Langmuir in 1916.e. The molecules i n this position stood upright with their polar heads touching the water surface while the non-po lar hydrocarbon chains lay parallel to each other at right angles to the water layer. By using one of the troughs. described earlier. the area of and the forces per u nit length are directly measurable. both with each other and with the molecules of the underlying liquid. . If the area available on the water surface to a cert ain number s of the film is reduced. Accordingly. He found the tension to increase markedly when ttl. the rela tionship film area (surface area} and film pressure {surface pressure} at a given tempera ture same dimensions of energy just as does the product of pressure and volume and ca n be to the thermal energy term nRT where 'n' is the number of moles. 'R' the gas con stant T the absolute temperature. the film pressure increases. A and B are movable bah'lets for cleann9 purpose.5 A per molecule by this technique. the molecules form a team and the system behaves differently fr om an independent molecule. for example. stearic acid was found to have a cross-sectional area of 20. various aspects of protein structure can be deduced by investigatio n of spread protein films. polysaccharides and with other interfacially-b ound compounds. i. 556/20. surface films have also been used to study the effect of irradiation of proteins and the results supplement the information obtained by m re conventional methods. In fact. it is a system whoseroperties are determined by an orderly arrangem ent of molecules. Tumit in 1954 has described a method for obtaining protein diffusion coefficient (D) by monolayer technique which is less cumbersome and is speedy too. i.e. Lastly.. it was possible to calculate the length of stearic acid molecule. F is thefloat. we have the realization of a simple molecular functi onal unit.. As for example. the proteins interact with lipid. In addition. the whole living body is a functional unit where varied molecules inter act and co operate with each other. Many immunological and enzymatic reactions have been studied by surface film tec hnique. the study of compressibility of a protein film permi ts the determination of the molecular weight of the protein molecules forming the film.1 The monolayer technique is extremely significant and has numerous applications. Such functional systems are often encountered in biological structures .168 Biophysical Chemistry Figure 7. Knowing the molecular volume of stearic acid (556 A). With a system such as a monolayer.15 Monolayer of stearlc acid molecules formed on water n a Langrnulr tr ough.e. Adsorption and Surface Tension : The Gibbs' Msorpflon Equation Surface tension is regarded as one of the different forms of energy acttfig at s urfaces giving .5 = 27. for example. In other words. C is the movable barrier used for compression ofthefdm. rise to adsorption. On the other hand. In the course of ordinary thermal agitation. As they concentrate in the surface film. In the process. the solute molecul es are brought to the surface. they are restricted in their movement because their esc ape would cause an increase in free energy and then work would have to be performed. But the com plete replacement of the solvent by solute molecules in the surface will be prevented by thermal agitation and forces of molecular attraction. Consider f or example a solution where the solute components exhibit positive surface activity (lowers s urface tension of the solvent). in a solution w here the solute . they lower the surface ten sion of the solvent. This is more evident at the liquid air interface. (4) Adsorption 169 component exhibits negative surface activity (increases surface tension of the solvent)... Gibbs in 1872 and later independently by J. The surface phase consists of those molecules which are close eno ugh to the surface to be acted upon fully by the surface forces. The solute molecules. no such of solute molecules in the surface layer is seen. free energy. in thi case. A surface phase L7 defined in. (//) the free energy of the solute (n°).follows. G°.. The i. The exact thermodynamic relationship between adsorption and surface tension was first derived by J. The area of this surface phase will be denoted A°. this manner has an area but no thickness. We shall designate the the rmodynamic properties of the surface phase by a symbol '' and place it as a superscript. In other words. It is known as the Gibbs' adsorption equation which can be derived as . and (///) the free energy o f the solvent (2n ).W.. We by are considering a L system at constant temperature and pressure and constant composition of the bu lk phase. (3) . This is a feature particularly observed with solutions of inorganic salts. of the surface layer is made up of three factors : (/) the fr ee energy due to surface Ltension (?A°). Let us consider a solution which has attained equilibrium between its bulk phase and surface phase. Thompson in 1888. DI and 2 are the chemical potentials of the solute and solv ent respectively while n and n2 Ddepict the number of moles of solute and solvent respectively. a re rejected from the surface and thus have to be content with remaining in the bulk of the soluti on..J. it is a st rictly twodimensional phase.. The superscripts indicate that . it can be brought ab out by (/) varying the surface area which will cause the change ?dA°.W the functions apply to the surface. Thus.dn' +n'dl +dn. (ii) varying the am ount of solute which will cause the change Bdn° or (//0 varying the amount of solvent. at constant temperature and pressure G = Eiani and the equation dG = ?dA i i! relating change in free energy with the surface tension (y) and change in sur face area (dA). r At constant temperature the equation becomes G = yA + Bn + B2n .. Thus the differential form of the surface free energy becomes dG° = ydAa + Bdn + l2dn2 But the complete differentil of G° is dG =ydA° +A*dy +t. which will cause the change B2dn°. for the surface phase we can write G° o I . Comparing Equation (3) and (4) we get .I [)\ ---S.. where S° is the surface entropy and T is temperature. Equation (1) can be obtained from the thermodynamic relationship at cons tant pressure . (2) If a change in free energy of the surface phase is desired. fin dt= RT dln a ... In such a case. Ci°.. If the concentration of the solute is small... a change in concentration will not affect the free energy of the solvent. (5) by A gives dy=-CIdBC2d2 The number of moles of solute present in the surface in excess of the number of moles of the same solute in the interior is taken to be the molar excess of solute. This molar excess of solute is the amount of solute adsorbed at the surface. (9) . (13) Biophysical Chemistry Now the surface concentration.P dt ..= o + RT In a where 'a' is the activity of the solute.... (6) . (7) . Then dy = . {io) . (8) where F is the concentration of the solute in the surface phase.170 . Since the chemi cal potential is related to the activity by . division of Eqn.... in moles per unit area is defined by Therefore... (11) . the excess C of the solvent vanishes. I is positive and the concentration of the solute in the surface is greater than the bulk phase. . I is negative and the body of the solution is richer in the solute than the surface. (12} RT dlnX and for dilute solutions where the activity is very nearly equal to the concentr ation.dy RT dlnC RT dC This expression is called the Gibbs' adsorption equation.. dy/dC is positive. and I dy Ci . We can also see from equation (13) that when the surface tens ion of a solution decreases with concentration. On the other hand. so that dy = .. the moIe. This is observed in solutions of many electrolytes. The above equation has also been experimentally verified by McBain and Humphrey. the molar concentration. They employed an a pparat-s .. we have dy = .Substituting equation (10) in equation (8).F RT din al For an ideal solution. The equation provides a mathematical basis for the generalization that if a substance tends to reduce su rface tension it will collect in the surface phase while those that tend to increase surface tens ion will be retained in-the bulk phase. when the surface tension of a solution inc reases with concentration. The surface active agents exhibit this type of behaviour. a = X.fractlon.F RT dln X 1 d7 or r . dy/dC is negative. X becomes I proportional to C. On the othe r hand.from an alcoholic one. solvent. When an adsorbent is dropped into a solution either the solute or the may become adsorbed. ADSORPTION FROM SOLUTIONS" A solution is made up of two basic components : solute. the mechanism of adsorption is by virtue of electrostatic forces of attrac tion. when the solvent is preference to the solute. The experimental F obtained satisfactory agreement with that calculated via the Gibbs' adsorption equation. When the solute is taken from the solution by an adsorbent. whether positive or negative. an d those which decrease interracil energy tend to concentrate at a liquid-solid or liquid interface". It follows from this principle that the lower the surface tension of the solvdnt. surface tension are the factors resjonsible for an increase or decrease in The Gibbs . Similarly.Adsorption 171 resembling a microtome to slice off the top layer (0.) of the surfa ce from a was later collected in a sample tube for analysis. Conversely. it is calledposit/ve adsorpt/or In this case the concentration the solution decreases after treatment with the adsorbing agen t. when a blue in water is shaken with solid carbon. activated carbon c an be used remove acetic acid from a solution of acetic acid in water. For example. is best from solvents of high surface tension. picric ac id is adsorbed upon charcoal from aqueous solution than. which may be one or more in and.Thompson princle states that "those substances which urface energy tend to concentrate at a liquid-air interface (in the surface).e. Adsorption from solution is very similar to adsorption of gases and is governed by the i. a part of the dye is removed by solid carbon and the solution becomes more dilute. When solvent and solute are adsorbed. The I adsorbed upon charcoal from water may be readily recovered by washing with alc ohol.05 mm approx. and so the decrease of the surface tension of the aqueous solution with concentration of picric acid is likely to be greater in aqueous than in alcoholi .. the less will be the amount of adsorption. The for this behaviour is that water has a much higher air-liquid surface tension has alcohol. the concentration of the solution after the ads orbent has will depend on the relative extent of adsorption. because here the solute causes th e decrease in surface energy at the solid-liquid interface. This phenomenon is known as negative adsorption. As an example. Adsorpti6n of the solvent is xery rare. the concentration of the solution actually increases after° with the adsorbing agent. conditions being uniform. these two components displace each other in the surface layer . The reason behind this bservation is that molecular a fundamental cause of adsorption. or powdered are raore effective in adsorbing non-eleCtrolytes from a solution than electroly tes. vapours. Rise of of adsorption. When various inorganic salts are dissolved in water.. in that the solution contain s two and solvent) forming a closely packed layer on the surface. The effect of temperature on adsorption equilibrium is worth mentioning. is disrupted by thermal agitation. a observed only during adsorption from solutions.in spite of the fact that surface tension with a rise in temperature. pure liquids. Therefore. Adsorption from solutions on the surface of solid adsorbents differs from the ad sorption of substances like gases. upon heating. the blue colour disappears due to the failure ofi0dine th remain on the starch particles at higher temperatures. same is observed at the interface of adsorbing solid and liquid solvent. iodine is added to starch solution. This is. etc. . Upon a change the concentration. neither the surface soluti on the bulk of the solution has any free sites and only displacement of molecules o f one by molecules of the other takes place. the surface tension of the solution is r increased. For exampl e. Thus. solid adsorbents like powdered silica.c solution. a blue colour appears due to formation of st arch However. activated carbon. 172 Biophysical Chemistry The amount adsorbed from solutions depends on the properties of the adsorbent. Practically. THE IMPORTANCE OF ADSORPTION PHENOMENA Adsorption is associated with many of the reactions taking place in the living b ody. thus speeding up many chelIfl'cal reactions. active mass and concentration are synonymous and an increase in concentration of the components of the reaction facilitates the reaction. industrial applications as well as various technical applications. being colloidal in nature. Polar molecules that are capable of formi ng H-bonds with the hydroxyl groups of the surface of the adsorbent are especially greatly adsor bed. Enzyme action also invo lves adsorption. This is clearly evident in the case of porous material like charc oal. alcohols. provided other factors are in optimum conditions. An increase in surface area of the adsorbent greatly enhances the adsorption of substances from solution. fuller's earth. t hose of the solution. In the following text we shall discuss some of the uses of adsorpt ion. and powdered substance like silica. Adsorp tion taking . The adsorption of organic compounds from solutions of highly p olar solvents on the surfaces of polar adsorbents is negligible but adsorption of such substan ces is strong on non-polar or weakly polar activated carbons. water and amines. Catalysis The velocity of any chemical reaction depends to a great extent on the active ma sses of the reactants. It is for this reason that silica which has a hydroxylated surface can smoothly adsorb phenols. By this process. substances ordinarily present in l ow concentration may have their effective concentrations lncreased tremendously by being accumula ted at the interfaces. The enzymes. In the reactions taking place in the protopla sm. and of its constituents. have large surfaces and surface adsorpti on of reactants is a requisite for the enzymatic reaction to proceed smoothly. adsorption plays a very important role. The colloidal mol ecules are interspersed with a network of membranes which possess enormous surfaces. It also has numerous laboratory applications. "ous drugs andpoisons. Macromolecular Association and Dissociation Various types of molecular associations seen in the living body are as a result of adsorption. absorption depends upon th e concentration of the molecules at the surface or interface. The rate of their entrance into c ells depends to some extent on their accumulation on cell surfaces prior to penetration. Selective adsorption of drugs may thus be responsible for specc action. where they serve to trace the pathway and distr ibution of dissolved substances across cells and tissues. To this class of surface-active agents belong varied.' are concentrated at the interfaces and exert thei effect from that location. The se substances. and proteins and salts continuously take place in the cll p rotoplasm and are vital for the structural integration and function of the living cell. proteins and carbohy drates. Action of Drugs and Poisons Substances capable of reducing interracial tension tend to accumulate at the int erfacesI. Acidic and basic dye s which can be distinguished by their staining power are important tools not only in histologic al studies. as consequence of reducing interracial tension. These molecular associations between proteins and proteins. The cel l colloids contain . dissociation of macromolecules could be dictated by their desorption behaviours. Histological Studies Though adsorption need not be followed by absorption. Likewi se.place at these membranes promotes several vital chemical reactions. proteins and lipids. but also in experimental physiology. hromatography One of the most impoFtant laboratory applications of adsorption is the recovery and of vitamins. These odours can be effectively removed t adsorption on charcoal. adsorbing agents have found wide use in industry and laboratory for puri fication .. Purifiers used in toilets. the details of which are discussed in one of the subsequent chapters. are generally adsorbents for gases which e asily remove the thus freshening the air. proteins. CO. hormone. i Charcoal can also be used to remove odours from gases. etc. Thus. title "Adsorption Chromatography". and other biological substans by the method of analysis. Gas are devices containing an adsorbent or a series of adsorbents which purify the a ir for r adsorbing the poisonous gases and vapours from the atmosphere. evolved durin g the process. in Research and Industry Lastly. refrigerators. carbonated beverages can be made fit for consump tion. in Safety Devices A bacterial toxin. or a mineral poison may be inactivated by adsorption . often has objectionable odours. Ferric a good adsorbent Is often used as an antidote in cases of poisoning by arsenic. Applications of adsorption from solution include the clarification of sugar liqu ors by removal of Impurities from petroleum oils and motor spirits by colloidal silica and of dyes from dilute solutions In a number of solvents.173 their large surface a number of cationic and anionic centers which are the sites of electrostatic of the basic and acidic molecules respectively. This elution is also employed in the separation and purification of plant alkaloids. These resins have also found use in med icine such purposes as reducing the acidity of gastric juice in cases of peptic ulcer. vitamins and substances which are difficult to Isolate by ordinary methods. . While vitamin B is mostly adsorbed. Elution of vitamin B from silica is carried out by using an alkaline sol ution For adsorption of positive and negative long from solutions. The impure solution contai ning enzyme at a parti. the adsorbent is washed and then the enzyme (desorbed) by washing the adsorbent with a solution of another pH. reduction of ' and K÷ concentration in body fluids in cases of congestive heart failure and ren al failure. For example.Adsorption has been used for purification of enzymes.cular pH is brought in contact with an adsorbent such as alum inimum After the enzyme has been adsorbed.. Under these conditions. I ndustrial have made good utilization of ion-exchange resins for water softening and purposes. ion exchange resins have used in the laboratories and industries. is not. removal of electrolytes from food material and other products and purification of metals. also serve to adsorb toxic products of bacterial action in cases of diarrhoea. vita min B vitamin B extracted from yeast are separated from each other by adsorbing vitami n B on gel in acid conditions (pH 3). D.J. New York. Chemisorptior Butterworths.. Gregg. 1967.174 Biophysical Chemistry Suggestions For Further Reading 1. Butterworths. 3. London. 4. P. Lond on.. Academic. 1957. (ed. Emulsion Science. Physical Chemistry fSurfaces.. A. 1967. and Sing.E. . 5.W. New Yo rk.. Surface Area and Porosity. New York. 1966. Academic. S. W. John Wiley and Sons Inc. Shaw.. Sherman. 2.).W. Adamson. Introduction to Colloid and Surface Chemistry. 1967. Garner. Adsorption. S.J. I {A) The electromagnetic wave. E Direction of propogation q 1 IIIII III I=:Jllll I I Magnetic Field. .1A).. (Figure 8. nanometers (nm). and/or its stru cture. Electric field. Figure 8-1A den otes one of plane polarized light. Thus. or units (/). a of light may be understood as an electromagnetic wave-form disturbance or photon propagated at 3. where Inm = 10-3 tm = 10-6 mm = 10.1A). IB denoting variatio n in the and magnetic field with respect to time at any given location in a plane polariz ed ray. micrometers (m). v. corpuscular and waveform. i. A beam of light from a bulb consists of many randomly oriented polarized components being propagated in the same direction. electromagne tic description of the radiation in that the radiation is made up of an electrical a magnetic wave which are in phase and perpendicular to each other and to the of propagation (Figure 8. may be measured in centimeters (cm). Wavelength ()) is the distanc e between two crests or troughs. In the following pages the ory. The term. 8.8 SPECTROPHOT OMETRY In this chapter an attempt has been made to describe certain relationships among optical phenomena which produce signals conveying chemical information either to the concentration of the chemical in a given solution. is used rather than wavelength to describe a par ticular To understand this term it will be helpful to see Figure 8. at the speed of light. and 1A = 10-8 cm. The magnitude of electrical vector is denoted by symbol E and that of magnetic vector is denoted by the symbol H.e. frequency.cm = 10-9 m. and applications of each of the optical signal sources has been discussed BASIC PRINCIPLES Light is supposed to have dual characteristics.0 × 108 m/sec. The magnetic vector (unshaded) and electric v ector (shaded) are perpendicular to each other and to the direction of propagation. The distance along the of propagation for one complete cycle is known as wavelength. information afforded by different optical signal source is so distinctive and va luable each has spawned its own specialized instrumentation. Sometimes a term. number of waves passing through a fixed point on the time axis per second is kno wn as the . v.176 Biophysical Chemistry frequency. Frequency shares an inverse relationship with the wave. ejection of an inner orbital electro . Oscillations of nuclei and electrons in electrical or magnetic fields. while the energy increases with the increase in frequency. 3 x 108 m/sec. Wave number means the number of complete cycles occurring per centimeter I)=11X The energy E. EIectrlc field. mo lecular bending and vibration. A beam of radiation from an electric bulb consists of several wavelengths and is known as polychromatic. E Magnetic Field. Electromagnetic radiation is produced by events at the molecular. atomic. the energy of the radiation decreases. length so that V = c/X where c is the velocity of light. A beam in which all the rays have the same wavelength is known as monochromatic. h c E=h=hm where c is the velocity of light in vacuum. Sometimes radiat ion. Remember that wavelength and frequen cy share an inverse relationship. this means that as the wavelength increases. is characterize d by another term known as the w<'e number and denoted by the symbol . of a photon can be related to its wavelength and frequency with th e help of Planck's constant. or level. excitation of orbital electrons. H Figure 8. I (B) The dependence of electric (shaded) and magnetic (unshaded} vect ors at a particular location tn a plane polarized electromagnetic wave. mostly in the infrared region (see later). of the radiation (usually expressed in Hertz or cycles per second) . The events leading to each component of this spectrum are also described in Figure 8. a complete spectrum o f electromagnetic radiation will be produced. . the radiation they emit will have different wavelengths.2. Thus. and nuclear break-up are some of the events which give r ise to electromagnetic radiation.n and rearrangement of the other electrons.2. Since each of these events differ in terms of the ene rgy involved. This spectrum is depicted in Figure 8. . cm Figure 8.1 2-180 1400-'780 ] 25.Nuclear transition X-rays Microwave Radio 177 3.01 0.000 nm .000 0. .the main regions and their wavelength s.125% Spectrophotometry Inner shell electronic transition Valence electron transition Molecular vibration & rotation Oscillations of nuclei and electrons in magnetic field Vacuum I I INear 0. Physical events involved in their production are also indicated.005-30 300 180-400 780-25.2 The electromagnetic spectrum -.000-125. If the intensity of the radiation incident upon such a thickness is assigned a value of 1.3 and may well ass ume the graphical Figure 8.e.5)2. This process is illustrated in Figure 8. 50% of 0.3 The pattern of light absorption by successive equal path-lengths (b) of absorbing solution.One of the earliest studied characteristics of chemical compounds was theicolor. THE LAWS OF ABSORPTION The absorption of light by any absorbing material is governed by two laws. (0. The substance in question here is absorbing most other component s of the electromagnetic spectrum except green. it will absorb 50% of the transmitted beam. The second transmitted bea m will then have a value of 0. If we now place a second equal thickn ess b. .5)I..5)3 etc. The successive light intensities are the sequence (0. That color intensity should form the basis of most widely used set of chemical assay procedures is then but natural.5. i.25. Things are colored because of their ability to absorb certain components of the electromagneticietrum with hich they interact.e. Consider a substance which ap pears green in color. he transmitted beam will have a value of 0. To understand the above statement let us assume that a thickness b has the ability to absorb 50% of the incident intensity of the light passing through it. This is clearly an exponential function and may be expressed as I e -kb Io I = the intensity of the transmitted light.. If one can calculate the amount of light absorbed by this substance. form as in Figure 8. The f irst of these laws is known as the Bouger-Lambert law.5. (0. It states that the amount of ligh t absorbed is proportional to the thickness of the absorbing material and is independent of th e intensity of the incident light.4. which is being reflected giving it a gree n appearance. one can arrive at a fair judgement about the concentration of the substance.0. i. the out coming. Io = the intensity of the incident light. b = the absorbing thickness. better known by the term path-length . The power t erm in the above relationship can be removed by converting to the logarithmic form. In I-. or In I--?-° = kb. Io I changing to common logarithms we get. and C = the concentration Of the absorbing material.= -kb. Thus.o -.3 expressed graphically by plotting % trmlsslon agal nt the path lenjth (thickness] and k = the linear absorption coefficient of the absorbing material.178 25. Io 2.= kb The second law of absorption is known as the Beer's law.4 Data of "igure 8. This can be mathematically expressed in the form of an equation similar to the one above.= k'C I where k' = absorptivity constant. 100% Transmission 50 2 Path length Figure 8.e. We can now combine the two equations for the Bouger-Lambert law and the Beer's l .303 log lo-. the concentration of absorbing solution.303 log.. 2. This law states that th e amount of light absorbed by a material is proportional to the number of absorbing molec ules i. The combined e quation is written as l°g. This combined law states that the amount of lig ht absorbed (absorbance or extinction) is proportional to the concentration of the absorbing substance and to the thickness of the absorbing material (path-length). It is . I/Io is known as the transmittance. The reverse. k and k' merge to become a single constant a (see Box 8.). Beer's law. the relationship between transmittance and sample concentration is a non-linear one. On the other hand. Absorbance shares a linear relationship with sample concentration. Here. T (the amount of llght which escapes absorption and is transmitted). The quantity Io/I is know n as the absorbance or the optical density (O.1). the Boug er-Beer /aw.D.o-Io = abC I This equation has been alternately referred to as the Beer-Lambert law.aw. or more simply. Percent. so A 2-1ogl In the equation . between percentage transmission and absorbance. transmittmce .Absorbance p'igure 8.5.5 The relationship. For this purpose we rewrite the equation so that A [absorbance) = log Io .Box 8.1 is :the Mo!ar abso Spectrophotometj 179 therefore easier to use absorbance as an index of sample concentration. This rel ationship between absorbance and transmittance is shown in Figure 8.log I but Io is always set at 100% and log 100 = 2. The two terms are mathematically commutable and so one can be calculated from th e other. 180 Biophysical Chemistry . In order to c ancel out the O. of the unknown x conc. scale. standards will be read .D. The bit differently. If the substance does not absorb in the visible region. subsequently. The whole process is described .D. is due to the absorbance due to the solvent and the reagents. of the unk nown. This O.D. of a standard O.182 Biophysical Chemistry Stating Beer's law a ional to the sample concentration e optical density of standard solution is ed from its optical density. observed will be due to the substance only. of the standard The standard chosen should have an optical density nearer to the O.D. Before we go ahead. since the O. we prepare what is known as th e blank. The solvent and the other reagents might also absorb in the region in which the absorbance d ue to the substance of interest is being measured. When. This reading is now m ade zero with the help of a knob in the instrument. To measure the O. we can say that optical density is proport If the path-length is constant.D.D. a particular reading is obtained on the O. contributed by the solvent and the reagents. It follows then. will then be a sum o f the individual absorbances due to the substance and the solvent and the reagents. it is necessary to instill the concept of what is known as t he "blank" or the *reference" solution. many times one adds other reagents so that the compound becomes colored and absorbs in the visible region. which consists of all reagents used in the solvent but not the substance of interest. When this solution is read in a spectrophotometer. The most accurate way of measuring the concentration in the unknown sample is th rough preparation of a calibration curve from a number of standards.D. The resulting O.D. the O. of any given substance. one dissol ves it in a solvent.D. that if th a known. then an unknown sample concentration can be calculat formula used for such calculations is concentration of the unknown O.D. due to other regents (which are added in equal quantities in all the standards and the unknown) has already been cancelled. in Box 8.2. . however. is not so severe as in such experiments. It is not n ecessary . laws of absorption apply to monochromatic radiation and The deviation from linearity in this case. From Beer's Law Following factors may cause deviations from Beer's law. The most common factor causing from linearity is the use of a band of wavelengths to measure absorbance in most In the strictest sense. Deviations from Beer's law occur usually when hlgh sample concentrations are being One effect of high concentration is that the molecules may dimerlze.183 Spectrophotometry A standard curve should always be prepared to check that Beer's law applies to t he analysis performed (in which case the curve would be straight). I. Other factors which cause deviations are given below. This is necessary since t here are factors that may cause deviations from linearity. 3. The spectral shift is normally due to polymerizaUon. Micelles are usually formed by those molecules whic h have one of the ends 'phillc' to the solvent while the other is 'phobic. called micelles. The intensity of the radiaUon reaching the.6. A particularly interesting . There Is JUSt one wavelengt h here where the molar absorpUon coefficient has not changed. I f the spectra differ.6 (B) Schematic representation.6) leadin g to a positive or negative deviaUon.184 . the absorpUon coefficient will also undergo a change (Figure 8.ntain more than 100 molecules per particle. In such a case a positive deviation will be seen.detector Is thus lessened. and discu ssion under 'Asoclatlon Colloids'). and scattering. The micelles are huge particles by comparison since each micell e may co. Positive deviation --Negative Concentration Wavelength Figure 8.c alled the IsobesUc point. The micelles cause appreciable scattering of radiation in the X-ray to visible region. Biophysical Chernistnj that the absorpUon spectra of the dimers are the same as that of the monomers. Over an extremely small concentration range known as the critical micelle concentration (cmc). Large aggregates then scatter light . At there will be negative deviaUon and at 3 there will be a posI Uve deviaUon. Box 3.' Examples are fatty acids. hospholipids. etc. High concenaUon may also lead to aggregaUon. ionic lipids. Thus two diffe rent phenomena are contributing to the reduction in the transmitted intensity m absorpUon. (A)A schematlc of absorptWn spectra of the same substance at two dif ferent concentrations Figure 8.of how negative .manif estaUon of this phenomenon occurs in the formaUon of micelles (see Chapter 3. This wavelength (2) is . certain kinds of solute molecules cooperatively associate i nto large particles. a deviation from linearity will result. on the other hand. Ife were to make a third series of measurements now using a large half-bandwidth filter (20 m) with a maximum of 52 0 nm. The calibration series for a red solution is measured using monochromatic light of wavelength 492 rim. on the flank.. the resulting calibratiu. The calibration plots obtained are two straight lines having different Slopes.and positive deviations appear on the chart Aggregation. Naturally. The most significant point to note here is about proteins. at the maximum. Let us see what this means. On e of the main causes here is imperfect rnonochromacy. Instrumentation limitations may also result in deviations from Beer's law. . High concentrations can also lead to chemical reactions which will lead to a cha nge in the chemical composition of the solution.e. . i. Proteins are known to denature at low concentrations and the den atured product has an absorption spectrum that is different from the native protein. can lead to electronic interactions that can eit her lessen or enhance the absorption coefficient. plot will be a curved line. and of 546 nm. 2. i..Deviations may also occur at low concentrations.e. and so on. will be. The reason is not far to seek.Spectrophotometry . 3/8. The series will not rise in an exponential manner as for the previous t wo individual 2 Thus absorbance plotted for these composite transmittances will give rise to a (Figure 8. 1/2. 185 This is only an apparent deviation. Let us say that a given unit of concentration has the. However. 1/4. rough than we have just assumed. It is not difficult to see that if the absorptions are plotted for both these series against increase in concentration. (B) sensitivity changes . Let us. assume that the filter is passing only two wavelengths.. the same concentration absorbs only 25% of the . .will not in straight lines though there are no other conditions for violation of the Beer 's law. For the it will be 3/4. One of th e wavelengths is the 'm for the sample and the other wavelength is at the periphery of its abs orption spectrum in the red region. When we think that there are many more wavelengths passing th. the following factors can cause deviatio ns from i Beer's law.wavelength at t he periphery. an d so on. abilit y to absorb 50% of the 'm" However.15" 0 2 4 6 8 Concentration Fgure 8. result will be a straight llne for each. operation {to be discussed later) tends to cancel out most of the random causes of Apart from the factors mentioned above. for t he sake of understanding. 3/16.6 (C) Effect of imperfect monochromacy imperfect monochr'omacy.. for the whole filter the compo site transmittance k i + 2. other indeterminate instrumentation variations which include (A) stray radiation reaching the detector.6 (C)). and power fluctuations of the radiation source and detector amplification syst em. 1/8. From our consideration of the Beer's law in the earlier pages we can say that th e transmittance series with every unit rise in concentraon for the . 0. it is easy to see why measurement using filters . change in temperature results in a change in the of solubility. The absorbance in is always different. This insistence is not aqueous as well as other solu/ons expand upon heating. and several is also reflected in the absorbance. . Thus. dissociation/association properties of the solute. Moreover.Effects Most experimental protocols insist that i heating is requied for color developme nt. absorbance measurements always be don e at a constant temperature. hydration. the be cooled to room temperature before its absorbance is read. 7.186 Bophyscal Chemistry Sample Instability Experimental protocols for absorbance measurements of a few substances insist th at the measurement be done within a very short time of color development. This pattern is known as an absorption spectrwn. In such cases the color could increase. An example of a relatively simple absorption spectrum is shown in Figure 8. ABSORPTION SPECTRUM The pattern of energy absorption by a substance when light of varying wavelength passes through it is uniquely characteristic of the substance. To establish the absorption spectrum for a given substance. The data so obtained is plotted in the form of a curve relating transmittance or optical density (the latter is preferred) to wavelengt h. Th e application of absorption spectra is not limited to the visible region of the spectrum but may be applied equally wen to characterization of the ultraviolet or infrared absorption of man y substances. Since the absorption spectrum of a substance is usually characteristi c for it. transmittancy or opt ical density of a particular concentration of the substance is measured at different waveleng ths (the path length is kept constant throughout}. One good exam ple is the Fiske Subbarow method of phosphate estimation by the ANSA method. it may serve for its identification and may furnish information of analytical value. fluorescent inten sity also reaches the detector. deviations occur because apart from the transmitted intensity. . generally more com plex curves are found. Fluorescence Some solutes fluoresce (drugs and drug intermediates often do). For such substan ces. Turbid solutions always end up giving higher absorbance than what is determined by the color. may change.some colored compounds are unstable and undergo changes within quite short times. Even mor e detrimental is the fact that for some such unstable compountis. or decrease. even the X. The reason for this insistence is simple -. The dotted//he cated the wave/ejth of maxm sens/ttvfor spectrophotometrc measurement s.1. The wavelength at which the compound absorbs most is known as its absorption maxima. Absorption curves of a particular substance vary along the absorbance axis with variations in the concentration of the substance.7). it will be observed that absorba nce at all . ).. they do not vary along the wavelength axi s (curve x and y in Figure 8.. It becomes clear from Figure 8. represents a concentration twice as much as that of x.7 A simpl/ged absorption spectnan curve.7 that although a particular su bstance absorbs at numerous wavelengths.5 Wawength (nm) Figure 8.0 Absorbance 0. there are regions where it absorbs more than in other r egions. If one increases the concentration of a substance. . 1. or n = 3 etc. but does not accept energy from radiations of other wavelengths? The answer is provided by the concept of quantized energy. So why is it substance'S accepts energy from radiation of wavelength 270-280 nm to catapult i ts levels. This is the reason why o ne chooses the absorbance of a compound at its )m. whe re n has of 1. in light of higher frequency quanta will have greater energy. Quantum t heory also that in order to be excited the electron will accept only that radiation which h as the . is Absorption Spectrum Specific for a Substance? How is it that a compound absorbs maximally at one wavelength and does not absor b at mother?. Qua ntum energy of light is not continuous. to become excited the electron has to Jump from n =1 to n = 2. during a quantitative experiment.2. J 0 13. etc. These energy levels are signified by n.3 etc. the highest sensitivity may be obtained at a wavelength on absorption maximum.n=l Energy.8(A)). The theor y of suggests that absorption o radiation means acceptance of energy embodied in the to catapult electrons of the absorbing substance to higher energy levels. At thi s maximum sensitivity will be obtained in a photometric measurement. but is concentrated into packets energy or quanta. the of m atom can exist only in certain orbitals which involve different electron en ergies. however. Quantum concept forbids intermediate energy levels like 1. Let us assume that a substance S absorbs maximally between 270 and 280 nm | does not absorb at all at wavelengths beyond 300 nm or below 270 nm.ground state 187 has increased.5 .2. lowest level being the ground state. Thus a knowledge of the absorption spectrum of both.6. as for example in a procedure where the absorbance curves for reagents and color ed overlap considerably. there is a greater change in absorbance per unit change . . Occasionally. the compound is essential for the proper selection of an optimum wavelength.. As per quantum theory . the lone electron of hydrogen can exist in six different energy levels (Figure 8 . In a low frequency (high wavelength) light each quantum will h ave a small T while.concentration at the wavelength of maximum absorption. 51 Excited states 5.0. Associate d energies for each level are whiten in Joules and V.energy to push it into a permitted energy level.217/i.3.76x10-19 8. hydrogen electron needs a partic ular which is provided only by radiation of the wavelength 1. eV 0 0.1.42 x I0-19 .85 2. to the energy level n = 2 from the level n = 1..40 t Absorption of radiation of wavelength 1217 X lifts electron from n = 1 to n = 2 21.43 x 10-19 .0.54 1.87 x 10-19 . Consequently n=5 n=4 n=3 Energy. .36 x 10-19 . In other words it can absorb wavelength or frequency which provides it with its exact requirement.8(A) Ground state and excited state energy levels of hydrogen atom. and molecular orbital s Type of bond Transition of lowest elergy Shapq of molecular orbitals Ground state Excited state g eon system perpendicular to the plane of the page.1). Quantum theory. The following types of orbitals are genera lly found in the ground states of organic molecules (see Figure 8. the multlplicity of energy levels available to electrons belonging to different atoms result in t he discrete bands to coalesce and thus. Bonding morbttals: These are extremely strong and constitute single bonds betwee n atoms. The electrons are not at all delocallzed and the distribution of electrons is cy lindrically symmetrical about the bond [Table 8. In complex molecule s. vibrational and rotational energy levels are also associated with each energy level of electronic transition. The very few energy levels available for its lone electron transitio n give a sharply defined absorption spectrum (line spectra) for this element. Moreover. Going by the example of hy drogen we can easily explain why the substance S does not absorb at 300 nm but absorbs at 280 nm. broad bands are observed. . thus. Hydrogen is a small atom.8(B) and Table 8. I).hydrogen absorbs light of this particulkr wavelength. Table 8. Let us now briefly deal with the types of electronic transitions which t. Excitation of an electron and its promotion to a higher energy level is also accompanied by changes in the vibrational and rotational levels.ake pla ce when a given substance absorbs light energy. which are nevertheless specific for the given molecule. explains why absorption spectrum is specific for a given substance . I A few examples of some bond types. This is an additional factor responsible for the broad bands of absorption spectrum of a given compoun d. transitions. its electrons are promgted from a bonding to an an tibonding orbital (higher energy sates). there is no corresponding antibonding orbital fo r them. These lo ne pairs are not involved in bonds (non-bonding) and thus retain their atomic character. nitrogen).-------n According to the molecular orbital theory. The antibonding orbital associated with the ¢ bond is calle d ¢" orbital and that associated with the g bond is known as the ' orbital. Since non-bonding electrons are not decfly involved in bonding. The occupied orbitals with highest energy in such molecules are those of lone pairs. All the above three types are illustrated beautifully by formaldehyde as shown b elow : H/ . Bonding -orbitals: These constitute multiple bonds between atoms and are based o n a combination of atomic p-orbitals. The . n-orbitals : Certain molecules contain heteroatoms (e.Note : Special distribution and sign of wave function of the molecular orbitals has also been provided. oxygen. The electrons are strongly delocalized and int eract with the surrounding environment with relative ease..g. when a molecule is excited by the abs orption of energy (UV or visible light). These transitions are shown by alkenes. Since these transitions are associated with a energy. and azo compounds etc. the entire path-length is to be evacuated. n ----> n° transitions usually have very high extinction coeffI = 104 . these transitions. n ----> =°. Anti-bonding electrons Anti-bonding electrons Non-bonding electrons Bonding electrons Bonding electrons Figure 8.Spectrophotometry 189 major electronic transitions within the ultraviolet a'Kd visible regions are: ---> o°. and n----> x°. n ---> a'.8(B). carbonyl cyanides. these transitions frequ ently appear shoulders on the long-wavelength side of an absorption spectrum. n ---> * requires the least amount of energy. The usual spectroscopic ues cannot be used below 200 nm because oxygen absorbs strongly at this range. alkyne s. Several molecules. T hus. These transitions are also shown in Fig ure 8.8(B). All the above orbitals with ordering of their energies relative to each othe r along with the transitions are shown in Figure 8. The with these transitions are given below in the decreasing order. These transitions take p lace in the centres of molecules. ether.8(B) Schematic rnolecular orbital energy level diagram depicting relati . including water. Even lower energy is required for ----> n" transitions. This type transition usually takes place in saturated compounds containing one heteroatom with pair of electrons. Therefore.105 M-cm-) ff not forbidden by spin or symmetry selection rules. they take place at comparatively longer wavelengths easily obtained in a spectrophotometer. do the normal Ultraviolet region (180-400 nm). Energy required for n ----> * transition is lower than that of ¢ ----> a° transition . a ----> o > n ---> o* > ---> '> n ---> " It is clear that the energy required for ----> ° transition is of a very high orde r and ' short wavelengths are absorbed for these transitions. t herefore. It is because of t his that below 200 nm is known as the vacuum ultraviolet region. saturated alcohols . and show absorption attributed to n * transitions. The organic compou nds which all the valence shell electrons are involved in formation of sigma bond. The transitions just discussed have been summarized in Table 8. it may be said that the absorption bands of almost all o rganic normally found in the near ultraviolet and visible regions are due to either n ---> " n ----> * transitions. . The n ° transitions are usual ly symmetrical considerations and therefore have extinction coefficients of the order ust 10.2 along with info rmation the region of the spectrum in which they absorb.ve energy levels and allowed By way of generalizing. have extinction generally in the magnitude range of 103 .104. whereas n ---> * transition which are rarely forbidden. One can distinguish between n ----> n* and n ------> n* t ransitions by fat the extinction co-efflcients of the peaks at Xm. ground state to an antlbonding orbital of higher energy. or sometlme near UV (e..2 Types of electronic transitions Description Region of electronic spectra From a bonding orbital in the. g* (between orhitals) Vacuum UV (e. but also on the nature of the solvent in whi ch they exist. From an orbital in the ground state to a very high energy orbital (towards ionization) Vacuum UV Lastly.g. the structural character and the position of absorption maxima depend no t only upon the structure of the compound.190 Table 8. .g.. methyl alcohol at 174 nm. From a nonbonding orbital to an antlbonding orbital of higher energy (a) n ----> * Near UV and visible (e. (a) o ----> (between orbitals) (b) .g.. and the relative orient ation of ne/ghboring absorbing groups.. the polarity of the solvent or of neighboring molecules. ethylene at 180 nm.g. The environmental factors that can cause detectable changes in the absorption sp ectra are pH. benzene at 230 nm). nltrosobutane at 665 nm). acetaldehyde at 293 nm. Far UV. ethane at 135 nm) UV (e. methyl iodide at 258 nm). Also n ote the ral change In the absorption spectra at reglons other than the absorption maxOn .5.7 for clear understanding).2 0.9.9" 0. 0.3 0.6 0.Effects due to pH : It is the pH of the solvent which determines the ionization state of a molecule (see Box 1. With a change in i onization 0.8 and Tabie 1. travlolet absorption spectra of 3'-CMP.7 0. 0.8. The nucleotlde was obtained by enzymatlc degradation of tRNA.4 0. See the difference between the absorption at neutral and acidic pH.I 0 / \ A 240 260 280 300 Wavelength (rim) Figure 8. is used s a solvenL Orientation effects: The best example to understand orientation effect is theDNA .lble region. So lid line represents absorption spectrum in water while the dashed llne corresponds to 20% ethylene glycol The transition here is n -. Given below are the absorption s pectra (Figure 8.9) of the pyrimidine nucle. The effect occurs because in a polynucleotide. Mark the differ ence in the absorption spectra of the same compound in the two states of ionization. Polarity effects : In general. The chromophore is dejlned as any isola ted covalently bonded group that shows a characteristic absorption in the ultraviolet or the vl s. Most of the nitro compounds are yellow in color. Non-polar solvent s. For this re ason polar solvents have been used to distinguish between n n" and ---> =" transitions. the absorption spectra may also change. the base s are in close proximity.and therefore the spectrum is shifted towards shorter wavelengths when water. The absorption coefficient decreases even further with a double-stran ded polynucleotide because in this structure the bases are arranged in an even more ordered manner. THE CHROMOPHORE CONCEPT The old definition of chromophore regards it as any system which is responsible for imparting color to the compound. The modem day definition of chromophor e.otide CMP at neutral and acidic pH. nitro group is a chromophore which imparts yellow color. Absorption coefficient of a single nucleotide is greater than when the nucleotides are arra nged in a singlestranded polynucleotide. on the other hand. produce no such change. Clearly. This is true for polar chromophores. 260 270 280 290 300 310 Wavelength (nm) Fure 8. however. Some of .10). akes use of the term in a broader scope.25O Spectrophotometnj 191 status. polar solvents shift the n =" transitions to shor ter wavelengths and ----> ° transitions to longer wavelengths as compared to the g as phase spectra (Figure 8. which is more polar.10. Effect of solvent polarlty on the absorption spectrum of tyroslne. . acetylenes and ethylenes (Table 8. (//) Chromophores containing both and n electrons and involved in ---> " and n--> n" transitions. For ex ample. esters.3 ).3). nitrfles and azo compounds (Table 8.the important chromophores are carbonyls. acids. nitrile group of ethyle nic or acetylenic groups. (1) Chromophores containlng electrons and involved in ---> " transitions. For example. Chromophores are known to be of twotypes. carbonyls. Auxochrome is thus also known as a color enhancer. OR. -NHR.YSuch an absorption shift is known as the red shift. absorption maximum. Hyperchromic effect : This effect signifies an increase in the intensity of the. -NH2.1 I}. I . Sometimes decreasing polarity of the solvent may also cause bathochromic shift Hyperchromic shift Hypsochromic Bathochrom/c Wavelength {rim) Figure 8. . Important examples are -OH.192 Btophystca/Chemfstnj Auxochrome Awcochromes are groups which by themselves do not act as chromophores but whose presence brings about a shift of the absorption band towards the red end of the spectrum (longer wavelength). Figure 8. Bathochrom/c sh/Jt : This shift is due to the presence of an auxochr0me by virtu e of which the absorption maximum shifts towards higher wavelengths. or the bathochromic shift. -NR2. This resul ts in a new chromophore which has a different absorption maximum and probably an enhancedext inction coefficient. See Figure 8. Four uch absorption and intensity shifts are known and are detailed below. Auxochrome exerts its effects by virtue of its abilit y to extend the conjugation of a chromophore by sharing of the non-bonding electrons. -SH etc. In many instances the absorption and absorbance change either due to interaction with an auxochrome or due to change of the solvent. This effect is mostly due to the presence of an auxochrome {Figure 8.11. or a change in the extinction coefficient to a higher value at the same absorption maximum. This shift is du e to removal of conjugation and a change in the polarity of the solvent due to which the abso rption maximum is shifted towards shorter wavelengths (b/ue shift).11 Different types of absorption and intensity shIjs Hypsochromic shift :This is opposite of the bathochromic shift. INSTRUMENTATION FOR UV-VISmLE AND INFRARED SPECTROPHOTOMETRY In order to obtain an absorption spectrum it is necessary to measure the absorba nce of a substance at a known series of wavelengths. This effect signifies that the intensity of the absorption maximum is lowered {Figure 8.Hypochromic effect : This is the opposite of hyperchromic effect and is caused d ue to introduction of groups which cause distortion in the geometry of absorbing molec ules.1 I}. The instruments that are used to stu dy the absdrption or emission of electromagnetic radiation as a function of wavelength are called . by electric heating serve as excellent radiant energy sources.12 Block diagram of a spectrometer . (///) transparent vessels {cuvettes) to' hold the sam ple.adiant Energy Sources Materials whlcL can be excited to h/gh energy states by a gh voltage electric di scharge or . However. they em/t radiation of characteristic energies cor responding to AE. if the instrument applies wavelengths only visible range). Some mat erials have numerous energy levels very close to each other. These materials would constitute an ideal source for absorption measurements if the intensity of all the wavelengths is alike. (tO a monochromator to break the polychromatic radiation into component or "bands" of wavelengths. inpractice. Both the systems. but the radiation produced is not as stable as t . Xenon lamp may also be used for ultraviolet radiation. are. Consequently the wavelengths of radiation era/tied by these substances take the form of a continuum of radiation over a ve ry broad region. but all represent variations of the block diagram in Figure 8. however. :consist of a pair o f electrodes enclosed in a glass tube provided with a qumz window. this is not so and so we h ave different sources for different regions of the spectrum. Sources of ultraviolet radton : Most commonly used sources of ultraviolet radiat ion are the hydrogen lamp and the deuterium lamp. the energy difference between the excited and the ground energy levels. Sample --. More or less similar optical principles are employed in these in struments. When a stabilized high voltage is applied they ernit radiation which is continuous in the region roughly between 180 and 350 nm. available commercially involve quite a bit of complex arrangements. The essential components of a spectrophotometer include: (/) a stable and cheap radiant source. Te glass tube is tflled wi th hydrogen or deuterium gas at low pressure.Detect°r Amplifier &1 Holder " [ Recorder Figure 8.Spectrophotometry 193 or spectrophotometers (colorimeters. (/v) a photosensitive detector and an associated readout system (meter or record er).12. some important differences in the specific components used in the various spectrum. As the electrons of these materials return to the/r ground state. It requires to be heated up to 1500°C before it emits radiation in the range of 0. a narrow band width will g o a long way in increasing the sensitivity of absorbance measurement. Narrow band widths are mad e possible by using wavelength selector(s). Sources of visible radon :'lngsten filament Imp is the most commonly used source for visible radiation. Moreover. emits radiation in the 1-40 region.he hydrogen ]amp. which prov/des more intense v/sible radiation is used in a small number of commercially available instruments. Thus. narrow band radiation will allow the resoluti on of absorption bands which are quite close to each other. Globar is more stable than the Nemst Glower. as pe/nted out earl/er in the chapter. However. arc. Carbon. absorption of nrrow band width will show greater adherence to Beer's law. The Globar consists o a s/I/con carbide rod which when hea ted to approxLrnate]y 1200°C. It is inexpensive and emits continuous radiation in the regio n between and 2500 nm.4-20 . Wavelength Selectors All the sources discussed so far em/t continuous radiation over w/de range of wa velengths. . the laws of absorption in the st rictest sense apply only to monochromatic radiation. Sources ofnfrared radton : Nemst Glower and Globar are the most satisfactory sou rces o infrared radiation. emst Glower employs a h ollow rod of zh-'con/um and yttrium. Therefore. The essential components of a monochromator are: (i) an entrance slit whlch admits p olychromatic light from the source. and (v) an exit slit which allows the monochromatic beam to escape. Most modern filter instruments. Gelatin filters are made of a layer of gelatin. Narrow slit widths isolate narrow bands. Filters resolve polychromatic light into a relatively wide bandwidth of about 40 nm and are used only in coldrimeters. use tinted-glass filters. all the components in a mo nochromator assembly must not absorb in the range of wavelengths which are to be studied. (tO a collimating device such as a lens or a mirror whlch collimates the polychromattc light on to the dispersion device. For obvious reasons. Monohromators : As the name suggests. prlsms and diffraction gratings are cons idered below... The effe ctive band width of the light emerging from the monochromator depends mostly upon the dispe rsing element (prism or diffraction grating) and the slit widths of both the entrance and the exit slits. (No a wavelength resolving devi ce like a prism or a grating which breaks the radiation into component wavelengths.. A monochromator employing a prism for dispersion is shown schematically in Figure 8. lailters : Filters operate by absorbing light in all other regions except for on e. a monochromator resolves polychromatic rad iation into Its individual wavelengths and isolates these wavelengths into very narrow bands. whlch they reflect.13. namely. (iv) a focussin g lens or a mirror. Since the effectiveness of the resolving element is of pri mary importance. however.Source Sample holder 194 Biophysical Chemistry ¢'fffYgl'¢tlg'ffi sel'ecfors are of'two types. lens lens " . . However. One disadvantage of glass filters is their low transmittance (5 -20%). The entire assembly is mounted in a light-tight box. colored with organic dy es and sealed between glass plates. the slit width also limits the radian t power which reaches the detector. the two kinds most widely used. filters and mon0chromators. . fused silica or quartz prism are u sed. The latter is preferred. and (it) the material of which It is made. Some examples are NaCI. Simple glass prisms are used for visible range. Two types of prisms.Entrance slit Exit slit MONOCHROMATOR l%3ure 8. For ultraviolet region silica. the shorter wavelengths are diffracted most. . namely 60° Cornu quartz prism and 30° Littrow prism are usually employed in commercial instruments. F1ourlte is used in vacuum ultraviolet range. The degree of dispersion by the prism depends upon (/) the apical angle of the prisln (usually 60°). CsBr. Ionic crystalline materials are used in the infrare d region. KBr.13 Pasta monochromator Prisrr" A prism disperses polychromatic light from the source into its constitue nt wavelengths by virtue of its ability to refract different wavelengths to a different extent. and the mixed crystalline material commonly called KRS-5. Since it disp erses the short wavelengths more and long wavelengths less. the wavelengths at the red end of th e spectrum are not fully resolved. This is a major disadvantag e of a prism. they are said to be crowded. These grooves are also known as lines. Standard path length of these cuvettes is usually 1 cm. The solutions are d ispensed in cells known as cuvettes. and infrared regions. cuvettes of path-length of I mm to 10 cm are available for special purposes. Sample Containers Samples to be studied in the ultraviolet or visible regionare usually gases or s olutions and are put in cells known as cuvettes. This arrangement allows resolution of a single wavelength. quartz or fused silica cells are used in this region. Since glass absorbs in the ultraviolet region. Cuvettes meant for the visible region are made up of either ordinary glass or sometimes quartz.14 Diffraction grating and the dispersion of polychromatic radiation Gratings : Gratings (Figure 8. Sometimes spectra of solids may be taken directly. with an evacuated cell as a reference. Very often.Spectrophotometry Figure 8. In addition they. Most of the spectrophotometric studies are made in solution. the monochromator consists of both. a prism and a grating. placed before the grating is known as the foreprism. A grating may have anywhere betwe en 600 to 2000 lines per mm on the surface depending on the region of the spectrum in whic h it is intended to operate it.14) are often used in the monochromators of spectrophotometers operating in ultraviolet. visible. Spectra of gases are taken using enclosed ce lls. For this purpose. The prism . The major advantage of diffraction grating monochromators is that th eir resolving power is far superior to that of prisms. The surface of the cuvettes must be kept scrupulously clean. Standard path-length of gas cells is usually 1 mm but cells with path-length of 0. . the solids are generally in the form of pel lets. It preselects a portion of the spe ctrum which is then allowed to be diffracted by the grating. The principle behind dispersion of radiation by a gratin g is that it resolves light into its component wavelength by virtue of constructive reinforcement and destructive interference of radiation reflected. The grating possesses a highly aluminized surface etched with a large number of parallel grooves which are equally spaced. The pellets are kept in pellet holders for absorption measurements. However.1 to 100 mm are available for special cases.yield a linear resolut ion of spectrum which is not possible when prisms are used. fingerprint smudges and traces ofprevi ous samples. Rinsing with water should normally clean quartz or glass c uvettes. as we have discussed earlier. However. The two sides of such ceils through which the light passes are precision ground and polished to be optically flat The other two sides are rough ground glass and the cell may be handled by these. The use of rectangular cuvettes in spectrophotometers effectively curtails the chances of d irt being transferred during handling. Since most of the spectrophotometric studies are made in solutions. might cause serious errors in quant itative measurements. If. however. one should also take into consideration the effect that the solvent may have on the . the solvents assume prime importance. sulfonic detergents or nitric acid may be used .by causing interference in the optical path. the dirt is abnormally tenacious. The most important factor in choosing the solvent is that the solvent should not absorb (opt/ca//y transparent) in the same region as the solute. isopropyl-alcohols. methyl-. Some oft en used solvents with their upper wavelength limit of absorption are given in Table 8.4 Upper wavelength absorption limits of some solvents Solvent Upper wavelength limit (nm) Water Ethanol Butanol l-Propanol Ethyl-ether Iso-octane Hexane Cyclohexane Acetonitrile Methanol Dichloromethane Chloroform Carbon tetrachloride Benzene Pyridine Acetone 205 210 210 210 210 210 210 210 210 215 . chloroform.4. Table 8. The solvents which can be used in the UV and visible region are water. ethyl-. hexane etc.196 Bophysical Chemistry absorption spectrum of the solute. from a few centimeters to several meters. The latter is achieved by multiple reflections wi thin the cell. or CaF2 plates . phototubes. KBr. (iii) long term stability. (ii) short r esponse time. Detection Devices Most detectors depend on the photoelectric effect. Liquids are studied as thin films or solutions between NaCI. Ultraviolet and visible radiation detectors : There are three basic kinds of det ectors in this region. Important requirements for a detector i nclude (i high sensitivity to allow the detection of low levels of radiant energy. and photomultiplier tubes. Photocells. Solids are examined i n the infrared as pressed KBr discs or as suspensions in high molecular weight liquids ("mulls" ). where incident light (photons ) liberates electrons from a metal or other material surface. however. These glass tubes possess NaCl. is the basis of detection for infrared radiat ion. (i) Photovoltaic or barrier layer cells : It employs semiconductor materials. KBr. The current is then proportional to the light intensity and therefore a measure of it. Consequently detection devices used for this region are different than those ope rating in the ultraviolet and visible regions. and (/v) an electronic signal which is easily amplified for typi cal readout apparatus. or CaF2 windows for the passage of infrared radiation.235 245 265 28O 3O5 330 Infrared gas cells are made up of glass. Semiconductors are crystalline. Some sort of external circuitr y collects these electrons and measures their number as current.005 to 1 mm. The distance between the plates (path length) is usually 0. Change in thermal energy. and the bonding electrons between the crystals o f some semiconductors can be knocked out of their positions by incident radiation. Alth ough a number . The cells have varying path lengt h. selenium) seleni um based photocells are most common.of materials are used in photocells (cadmium sulphide. A typical photocell consists of a thin coating of se lenium over a . silicon. . This arrangement ensures that elec trons pass easily from selenium to silver but not in the reverse direction.ns to move away from the silver film.Selenium Anode Cathode Recorder Spectrophotometry 197 thin transparent silver film on a steel base. (B) a semi-eylindrical cathode whose inner surface is coated with alkali or alkaline earth oxide. and (C) a centrally locat ed metal wire mode.15 Selenium based photovoltalc cell Photocells have a long life and are inexpensive and reliable. A schematic dia gram of a phototube and the associated circuitry is provided in Figure 8. Due to the inab ility of electro . The current flowing between the two electrodes is then measured by a microammeter. Silver Steel back-plate Figure 8. The quartz window allows the passage of radiation which strikes the photoemissive su rface of the cathode. If the electron collection is 100% efficient. A representative diagram of the cell is given in Figure 8. A potential difference of approximately 90 volts is applied across the ele ctrodes. the silver acts as the collecting electrode for electrons liberated from selenium by the incident radiation. The steel plate functions as the other electrode.15. The electrons become excited and finally leave the surface and travel t owards the anode causing current to flow in the circuit. (ll) Phototubes or photoemiasive tubes : The components Of a phototube include(A } an evacuated glass envelope (with a quartz window). the phototube current should be proportional to the light intensity.16. They are widely us ed in colorimeters but their use in spectrophotometers is becoming limited. The energy of the photon is transferred to the loosely bound electrons of the cathode surface. (|iI) Pht.This is usually acc omplished by placing a high resistance (R in Figure 8. and (B) ad ditional .multl[r : These detectors are designed to amplify the initial photoele ctric effect and are suitable for use at very low light intensities. A photomultiplier consists of (A) an evacuated glass tube into whlch are sealed the cathode and the anode.Evacuated glass envelope I Anode Quartz Cathode window lgure 8.16 Dlram of a photoemislve the.16) in the phototube circuit. R stands for res/stance Phototube currents are quite small and require amplification. A photodiode array is a two-dimensional matrix composed of hundreds of thin semiconductors spaced very closely together.17 A photomultiplier tube (iVl Phottditdes : Photodlodes are semiconductors that change their charged volt age (usually 5 VI upon being struck by Light. The voltage change is converted to cur rent and is measured. At higher light intensities. Each diode is scanned. photomultip liers exhibit great instability. due to their great amplification power. Light from the instrument is disper sed by either a grating or a prism onto the photodiode array. and the resultant electronic change is calculated to be proportional to absorption. As the radiation strikes the photocathode. Photocathode Anode High voltage Figure.17 . In practice. photomultipliers are the d etectors of choice in all modern spectrophotqmeters. electrons are liberated and the applied potential difference accelerates the electrons towards the first dynode. Each position of diode on the ar ray is calibrated to correspond to a specific wavelength. Inspire of this tendency to be unstable. The liberated electrons are dragged onto the next dynode wh ere more electrons are released and this process goes on as a cascade till the last dynod e. By the time the electrons arrive at the collecting anode. Each su ccessive dynode is at a higher electrical potential and thus acts as an amplification stage for the original photon. The applied voltage causes sufficient electron acceleration to knock out other e lectrons from each dynode surface. the initial photoelectric current is a mplified by a factor of approximately 106.Incident radiation 198 Biophysical Chemtstr intervening electrodes known as dynodes. The arrangement is shown in Figure 8. The external circuitry is arranged so that a high voltage (1000 volts) exists between the ano de and the cathode. photomultiplier tubes are used only for low l ight intensities. The entire spectrum is es . 8. rmocoupl¢ is usually enclosed in a shielded evacuated housing. or lead teLlurlde). Consequently. Middle and far infrared detectors : When middle and far infrared photons are abs orbed. To av oid heat loss.sentially recorded within milliseconds. This preven ts error causing temperature fluctuations. resistance thermometers (bolo meters).8.0 . the electrons of the semiconductor are raised to conduction bands. their energies are convened to thermal energy leading to a rise in temperature. . lead sulphide. This causes a drop in electrical resistance. and gas thermometers (pneumatic or Golay ceils) are used as detectors in this re gion. Obviously then. Near infrared detectors : These are usually photoconductive ceLls which detect i nfrared radiation in the range 0.3. the the. if a small voltage is applied. Thermocouples used in the infrared receivers typically consist of a blackened go ld lead-teLlurium metal pin junction which develops a voltage that is temperature dependent. The sensing element is a semiconductor (german ium. Upon illumination with radiation of appropria te wavelength. The resistance of the system is such that the current may be amplified and finally indicated on a meter is recorded. a large incr ease in current can be noted. rapid response thermometers such as thermocouples. Visible Glass. ammeters. .nfrared spectrophoometers have been summarized in Tables 8. Table 8. This is accomplished by using amplifiers. potentiometers.5 and 8. or a foreprism grating double monochromator. Components of UV-visible and i. These signals need to be translated into a form that is easy to interpret . and potentiometric recorders. Detector Wave number (cm-1) Source of radiation Spectrophotometry 199 Amplification and Readout Radiation detectors generate electronic signals which are proportional to the tr ansmitted light. Quartz or fused silica.6 respectively.5 A summary of components of spectrophotometers and colorimeters Region of electromagnetic spectrum Ultraviolet Radiation source Optical system Material used in the Optical system Hydrogen or deuterium lamp Prism or diffraction grating. Tungsten filament lamp. Optical system Quartz prisms or prism grating double monochromator Diffraction gratings -10 . Photovoltaic cell. Detector Photomultiplier Table 8.6 A summary of the components of infrared spectrophotometers Region of electromagnetic spectrum Near-Infrared Mid-Infrared Far-Infrared 12. Tungsten filament lamp Coil of Nichrome wire. Round glass cells. or Nernst Glower.500. or Globar. High pressure mercury arc lamp.carbon arc (less used) Tinted glass filters or interference filters Sample holders Quartz or fused silica. 4000 200 . rectangular cells. Pneumatic or Golay mistor. KBr. Thermocouple. Lead sulphide. or pyroele. diffraction gratings. pr bolometers. ther. . ctric. or lead telluride photoconductive cells.with a fore-prism monochromator.cells. Optical elements are made up of ionic crystalline materials like NaCI. or KRS-5. CsBr. . double beam sp ectrophotometers have been designed (Figure 8. The two transm itted beams are then compared either continuously or alternately several times in a second. The double beam device. One of the split beams passes thro ugh the "blank" or reference cell while the other passes through the sample cell. The modifications described above make the double beam devices mo re sophisticated mechanically and electronicaliy as compared to the single beam dev ices. A rotaW sector chop .lflecUng sector 200 Bphysol Chemtry Double Beam Operation Voltage fluctuations inducing fluctuations in the source intensity can cause lar ge scale errors in spectrophotometer operation. and amplifier gain by observing the differences in signal between reference and samp le at virtually the same time. the double beam devices are expensive. Obviously. Mirror Blank I I Mi°ulflplie ono Chopper ple Open sector Rotating sector chopper Figure 8.. therefore..18 Optical arrangement of a double-beam instrument. Mirror . Double beam instruments employ som e type of beam splitter prior to the sample containers.18}. the det ector signal. compensates for fluctuations in the source intensity.. To obviate this situation. . a calcium chelator. in many reaction kineti c studies it is necessary to monitor the absorbance changes of two chemical species simultaneous ly. the ratio of the two absorbances can provide an idea of the calc ium concentration in the given biological system. Dual Wavelength Spectrophotometer Some metal chelators absorb at One wavelength before chelating the metal ion and absorb at a completely different wavelength after the chelation has taken place. An exa mple is that of arsenazo III. Similarly. The chopped be ams reach sample and reference and subsequently to the detector at intervals which depend upon the rotational frequency of the chopper. The device then records the ratio of the re ference and sample signals. 685 nm. The beam splitting usually occurs after the monochromator.18). If this chelator is incubated with a biological system and the absorbance of the chelator is measured simultaneously at the wave length pair 675 nm. This chelator absorbs at 675 nm before binding to calcium and at 685 nm after the binding has taken place.per s also shown. Rotatingsector mirror s are commonly used for splitting or *chopping" the beam (Figure 8. In many experiments it is necessary to measure the relative absorbances of proteins {280 nm} and nucleic acids (254 nm} simultaneously. For all these experiments it is necessary to use dual wavelength spectrophotometer. only a fe w of which are summarized below.UV-VIS SPECTROPHOTOMETRY Photometry being a very versatile technique. Table 8 .19 Optical arrangement of a dual wavelength spectrophotometer From the above it is clear that dual wavelength spectrophotometry provides infor mation from two wavelengths per unit time. is used. APPLICATIONS OF. This is usually done by plotting abso rption spectrum curves. Subsequently the two beams of d ifferent wavelengths are made to pass through the same sample by a complex arrangement of a large num ber of mirrors (Figure 8. Since these curves are specific for a class of compounds. has diverse applications. All other factors being equal. which is always a photomultiplier tube.Z2Cuvette Spectrophotometry 201 Dual wavelength spectrophotometry refers to the photometric measurement of a mat erial by passing radiation of two different wavelengths through the same sample before reaching the detector. the resultant data should be more useful than data from a double beam spectrophotometer. Qualitative Analysis Visible and ultraviolet spectra may be used to identify classes of compounds in both the pure state and in biological preparations.19). Only a single detector. Light from two different sources is allowed to be resolved into two di fferent wavelength with the help of a pair of diffraction gratings. Light source Mono chromator Monochromator Figure 8. a knowledge of the absorption spectrum can help in identification of a substance in biological milieu. If the .. compounds which do not absorb in 220-280 nm region are usually aliphatic o r alicyclic hydrocarbons or their derivatives. Absorption by a compound in different regions gives some hints of its structure. Sometimes they might be simple olefinic compo unds. It is quite beyond the scope of this book to deal with the details of identifica tion of an unknown compound or the assignment of structure on the basis of its absorption s pectrum (for details the student is referred to literature cited at the end of this chapter). Before attempting to interpret the absorption spectrum o f a given compound. Thus. suffioient chemical information about the substance such as the elemen ts present should be known.3 provides information about the absorption ranges of the most commonly occurring functiona l groups of biomolecules. a brief discussion is possible. However. one can tentat ively identify GMP from CMP since their absorption maxima are quite removed from each other. The maxima around 250-330 nm are characteristic ofa naphthaquinone. and UMP in aqueous solution at neutral pH. As an example to prove how valuable absorption data can be. However. With just these absorption maxima. Absorption in this range could also be due to benzene derivative s. it would be difficult to distinguish between AMP and UMP owing to their absorption spectra being quite close. t he absorption maximum at highest wavelength for GMP occurs at 255 nm. one can cite the exa mple of vitamin K whose structure was determined by the use of its spectral data. The ab sorption spectrum of vitamin K has the following absorption maxima: 249 nm. for CMP at 271 nm. Abetter example. CMP. Thus. a precursor of vitamin A has eleven double bonds in a conjugated system and appears yellow because the light in visible region (450-500 nm) is being absorbed by it. it will usually contain two unsaturat ed linkages in conjugation. 4-naphthaquinone. one can resort to changes in pH of these sol utions and the data so obtained can help us in identification. and for AMP at 259 nm.3 for further discussion. each of them possessing a characteristic shape and r ange indicating the presence of the particular functional group. A look at Figure 8. and 3 25 nm. If you dissolve the four nucleotides GMP. Complex systems will give rise to a bsorption curves with several maxima. 260 nm. However. Presence of more than two conjugated double bonds usually gives rise to absorption in the ra nge 250-330 nm. AMP. The structure of vitamin K determined later is as below: Also see Box 8. .20 and the te xt therein will explain the point. for U MP at 262 nm. Chemical man ipulation and comparison of K spectra with several model compounds indicated that the posi tions of the absorption maxima were similar to those of 2.3-dialkyl.202 Biophysical Chemistry compound absorbs between 220-250 nm range.20). It is a fact that as the number of conjugated double bonds increases. the ab sorption range is shifted more and more to higher wavelengths.1. is afforded by the four nucleotides (Figure 8. perhaps. -carotene. .¢ .'0 pounds o .7 6"0 .'O c"0 9"0 L'O g'O wv .t. 204 Biophysical Chemistry . can you answer the followingquestions? .0.zero fthe pH is now raisedbeyondt 0. only'three tryptophan residues seem to be on the surface. Solvent of lysozyme. text. f only two tyrosines an. it might quite frequ spectral changes are obtained with other stud es such as the so vent quite a bit of information about the structure of the active stem ght be studies. If the solution of the enzyme with the substrate is again studied by solvent perturbati on method.. one observes maximum of tryptophan is shifted to a longer wavelength.(.205 rise? behavior of Spectrophotometry be .¢ rise in absorption at295 nm will be seen because these tyros nes w I be sh elded . The conclusion is quit e Obvious really. all tyrosines wi be available for titri0n becausethe protein is now denatured. riein absorpt on at 29 5nm w I only be hail of what is expected for four tyrosines. Inthe last Case if the pH is rai sed tosay 13. largescale unfold ng of the protein were buried when the protein was in the surface brings about. It seems that the e.nzyme has one tryptophan residue in the active site which might be involved in the binding. what do you are rosine. pectral change corresponds with only one tryptophan residue. Studies with x-ray diffraction confirm this conclusion. On the surface and two buried in the folds. a change n the proteins: Whenever the substrate orthe competitive inhibitor region or makes certain aminc acid residues In doing so. (a) A protein solution is being heated and its spectral characteristics are being determined at 250 nm (. What happened? .Tle ext nction at 260 nm shows sudden drop. for cysteine). What conclusion should you draw? (b) A DNAsolution is being cooled after it was heated.. At:45oc the extinction of the system suddenly increase s.. This increase plateaus Out at 55oc. Choose a peak that is as far as possible from the absorption peaks of co mmonly interfering chromogens. What do we mean by selecting a suitable wavelength or alsorption band? There are certain rules for the choice. These may be summarized as under (Figure 8. the absorption spectrum of the chemical spe cie can be dctermcd experimentally by means of a scanning double beam spectro-photometer. 2. All proteins will therefore have a dif ferent absorbance at 280 nm and may be only accurately assayed if a calibration curve {see Box 8. The absorbance at 280 um by protein s depends on their tyrosine and tryptophan content. Nucleic acids at 254 r un and prote/n at 280 nm provide good examples of such use. If the chemical species of interest has already been researched upon.ultravlolet/vlsible absorption spectrum would be avai lable in the literature. Absorptivity at any given wavelength is constant and is an inherent characterist ic of the absorbing substance. Thus. Choose an absorption peak with the greatest possible molar absorptivity. its . 1. Choose a relatively broad peak. the first step is the choice of the absorpti on band at which the absorbance measurements are to be made. If this is not the case.21.Wavelength 206 Bophystcal Chemistry Quantitative Analysis In developing a quantitative method for determining an unknown concentration of a given species by absorption spectrometry. The same is true for solvents and other reagents used the absorption band chosen should be as far away as possible from the absorption pea ks of the solvent and the reagents. Choice of the absorption band for quantitative analysts.2 } is plotted for the pure protein. a number oflmportant classes of biological compounds may be measured semi-quantitatively using UV-visible spectro-photometers. 3. . Figure 8. A suitable absorption band is then selected from within the absorption spectrum for quantitative measu rements.21). Most of the organic compounds of biological interest absorb in the UV -visible range of the spectrum. ) and the peak is broader than . k3 then becomes the best choice. X has the highest absorption coefficient but the peak is too sharp.X has a small absorption coefficient. '3 has a sufficiently high absorption coefficient (though lower than . Of course. . Quantitative analysis should normally not be carried out here. this must not correspond to absorption peaks of other interfering chromogens or the solvent. . No other component of the reaction. simplicity and convenience of optical assays prompts their use in following the time of an enzymatic reaction in which neither the substrate(s) nor product(s) have a ny absorption maxima. either substrate or tbsorbs at 340 rim. Lactate + NAD÷ . and a proto n. does not. which have the common intermediate pyruvate: . Phosphoenolpyruvate + ADP v. An example of the reaction hosphoenolpyruvate and ADP yielding pyruvate and ATP catalyzed by pyruvate kinas e below. Example can be cited of the measurement of the enzyme lactate dehydrogenase.207 ay The quantitative assay of enzyme activity is carried out most quickly and conven iently the substrate or the product is colored or absorbs light in the ultraviolet rang e because or disappearance of a light absorbing product or substrate can be followed which gives a continuous record of the progress of the reaction on a chart recorder. Such type of enzyme reactions are coupled to some other reaction which has an easily measured optical change. NADH. NADH. The products of the reaction are pyruvate.Pyruvate + ATP Although neither the substrates nor products of this reaction absorb light in th e 300-400 the reaction is easily measured if lactate dehydrogenase and NADH are added to t he large excess. By manipulating the system in such a manner we obtain the followin g I reactions. It is thus very obvious that the progress of the reaction in the llrection can be followed by measuring the increment in light absorption of the system nm in a spectrophotometer. which is engaged in the transfer of electro ns lactate to NAD*. absorbs radiation in the ultraviolet range at 340 nm whil e its NAD÷. Other light absorbing or light scattering substances must either be absent by appropriate blank measurements.Pyruvate + NADH + H÷ One of products. is different for compounds of different molecular weights. M = awb/OD or M= 10a/E IL . Such measurements are known as assays and are fairly routinely used. Although the extinc tion of the absorption band remains constant in all the derivatives. M. a molecule of NADH is oxidized to NAD÷ in the second reaction when the converts pyruvate to lactate. of the be readily calculated on the basis of its absorption data. the optical dens ity. Weight Determination If a compound forms a derivative ¢ith a reagent which has a characteristic absorpt ion ' at a wavelength where the compound does not absorb. Since NAD÷ does not absorb at 340 nm the absorbance on decreasing with increased pyruvate generation.Lactate + NAD ÷ Since we have added a large excess of NADH to the system. then the extinction f the derivative is usually the same as that of the reagent. the system now absorbs at 340 But from the reactions written above it is clear that for each molecule of pyruv ate formed first reaction.Phosphoenolpyruvate + ADP Pyruvate + ATP Pyruvate + NA{)H + H÷ -. The molecular weight. determination of empirical formulas. spectrophotometry (UV-VIS) has been used to study such physicoch emical phenomena as heats of formation of molecular addition compounds and complexes in solution.208 Biophysical Chemistry where w is the weight of the compound in grams per litre. The trans-isomer is usually more elongated than its cis counterpart. Impurities i n a compound can be detected very easily by spectrophotometric studies by experiment ally verifying whether the given compound shows an absorption maxima not characteristic of it. and diss ociation constants of acids and bases. Control of Purification This is one of the most important uses of UV-VIS spectrophotometry. Similarly benzene impurity in commercial absolute alcohol can be detected by measuring absorbance at 280 nm where alcohol (210 nm) . The method has an accuracy of+ 2%. determination of reaction rates. Molecula r weights i of only small molecules may be determined by this method'. hydration equilibria of carbonyl compounds. hloride can be detected easily by measurin g absorbance at 318 nm where carbon disulphide absorbs. Thus carbon disulphide impurity in carbon tetrac. Study of Cis-Trans Isomerism Sine geometrical isomers differ in spatial arrangement of groups ab.out a plane. formation constants of complexes in solutio n. Other Physicochemical Studies Over the years. chlorophyllprotein complexes. the absorption spectra of the isomers also differs. Molecular weights of amine picrates. determination of labile intermediates. for the trans-isomer to have a highe r wavelength of maximum absorption and also to have a higher ema' Absorption spectrometry can th us be utilized (indeed it has been) to study cis-trans isomerism. association constants of weak acids and bases in organic solvents. vitamin A aldehyde-protein complex. protein-dye interac tions.. sugars. and many aldehyde and ketone compou nds have been determined by this method. tautomeric equilibria involving acid base systems. and b is the pathlengt h. association of cyanine-dy es. It is usual therefore. the liquid in this system might absorb at a particular wavelength. Turbiflimetry and Nepheloraetry Bacterial Or any other particulate suspension makes the liquid turbid.does not absorb. Example can be cited of barbiturates which show characteristic chan ges in absorption spectra between their keto and enol forms. The common features in the spectra cancel out and the bands which are recorded can be interpreted in terms of known differences between the sample s. This is d ue to Tyndall effect which addresses itself to light scattering by colloidal particles (see chapter 3). D ifference spectroscopy involves comparison of absorption spectra of two samples which diff er only slightly in their physical states. Moreover. It may also be used to demonstrate ionization of a chromophore leading to identification and quantitation of various components in a mixture. Difference spectroscopy has also been utilized in toxicology laboratories for an alysis of many toxic drugs.ary tool to study globular protein conformation. Difference spectroscopy was developed by Chance and Williams in course of their research on electron transport chain proteins in the mitochondria. difference spectroscopy is a necess. While. Difference Spectroscopy Difference spectroscopy provides a sensitive method for detecting small changes in the environment of a chromophore. The technique subseque ntly provided much needed information about the state and sequence of the electron transport p roteins. A lot many commercial solutions are routinely tested for purity spectrop hotometrically. the pa rticles scatter . The scattered intensity is usually measured at right angles to the d irection of incident light. If. radiation of a wavelength which is not absorbed by the liquid is made to pass through this suspension.22(A).22(B). The wavelength used for this purpose is 600 nm. The principle of nephelometry is diagrammatically shown in Fi gure 8. then. is very tedious to standardize as the particle size is critical for accuracy (larger par ticles scatter more light as compared to smaller particles. Nephelometry is a term many times used synonymously with turbidimetry probably b ecause the two techniques are based on a common principle. The technique. This technique is routinely used to measure the number of bacteria i n a given suspension. Using this technique. ed ligh t will allow one to have an idea of the number of particles in the suspension. thus contamination of small particle sus pension by a small number of large particles will give a value far in excess of the number of small particles actually present). the latter measure the intensity of the light scattered by the particles in suspension.Photocell . known as turb/d/metry. It is also used for waste-water analysis as well as in the beverages and pharmaceutical ind ustries to evaluate the amount of haze present in the preparations. one can arrive at a fair approximation of the number of particles in a given suspension.Spectrophotometry 209 the incident light. For low concentration this method is more sensitive since zero c oncentration is represented by a dark background. On the other hand zero concentration in turbid imetry means full illumination. howev er. the apparent absorption will be solely due to light scattering by the particles. Nephelometry is commonly used for estimating the concentration of microorganisms . The light transmitted by the suspension will have lesser inten sity than the light which was incident. Measurement of the intensity of this transmiR. The major difference between turbidimetry and nephelometry is that while the former measures the intensity of transmitted light coming out of a suspension. The principle of turbidimetry is shown diagrammatically in Fi gure 8. Sample cell containing suspension ] [ (A) f . .° o Light Slit . rather.22 Principle of turbidimetry and nephelometry. (B) Nepheiometry measures the intensity of scattered light rather than transmitt ed light. electromagnetic radiation of this region has considerably lower energy. A turbidimeter measures the intensity of this transmitted li ght and a calibration cw' beaueen transmitted intensity and particle concentration can be drawn. THEORY AND APPLICATIONS OF INFRARED SPECTROSCOPY As evident from the electromagnetic spectrum diagram. (A) Light is scattered by particles in the suspension in sample cell. Consequ ently. infrared radiation is of m uch higher wavelength as compared to the ultraviolet and the visible region. it is associated with vibrational transitions of molecules as we will see below. Infrared radiation is. Lens source (B) I Lens Photocell detector Figure 8. The transm itted light is therefore of a weaker intensity. therefore. not associated with electronic transitions. Consequently. These vibrations are known as stretching vibrations (Figure 8.23{A)).210 In-plane deformations Scissor and Rock " N F Symmetrlc Antlsymmetrlc DF IN Out-of-plane deformations Twist and Wag Figure 8. Vibrational transitions are low energy transitions and these ene rgy levels correspond to the energies of electromagnetic radiation in the infrared region o f the spectrum. Such variations i n bond angles may be about ±0. Cslculatlon of Vibrational Frequencies The vibrational frequency of a bond can be calculated with the help of Hooke's l . The other type of molecular vibration.23 Types of vibrations : (A) Stretching.23(B)) involves cha nges in the positions of the atoms with respect to the original bond axis. infrared spectra are typically presented as percent transmission (transmittance x 100) versus wave number. the atoms rema in in the same bond axis.5A. The bond distances between the atoms in a molecule fluctuate to about ±0.5°. The presentation o f infrared spectra differs from UV-visible spectra in that wave number is used in this region rathe r than wavelength. accompanied by promotion of the molecule to an excited vibrational state. (B) Bending All the molecules are continually vibrating. known as bending vibration (Figure 8. a molecule can absorb infrared radiation of an appropriate frequen cy. These vibrations are of two types. It should be r emembered that while this increase or decrease in bond length is occurring. . +rni) where v is the frequency. the reduced mass of the system. mass 2n mim2/(m. The quantity mrn/(m+ m2) is often expr essed as m.0 x 10Sgs-2 mass of carbon atom = 20 x 10-24. k is the force constant of the bond and m and m2 are t he masses of the two atoms involved in bond formation.g . Let's try and calculate the approximate frequency of the C--H stretching vibrati on from the data given below.. k=5.aw which correlates frequency with bond strength and atomic masses. V°¢l Or V-. For example. Given below are a few bonds. This we predicted on the basis of the bond strength. the frequency should increase. On the basis of mass we may hazard a guess that C--H should absorb at a higher frequency as compared to CmC since the latter has a higher re duced mass. This simple observation tells us that the vibration of any given bond will be affected by th e other atoms and their bonds that it coexists with.211 Specrophotorrtr 5.3x10asDividing the above value with the speed of light we get the value in wave number which is 3100 cm-L When you experimentally determine the vibrational frequency for C--H s tretching you find it ranging from 2800 to 3100 depending on the compound you choose. If you look at the above equation. we are stepping onto treacherous territory if we start predicting frequ encies on the basis of masses without knowing the force constant. O--H. Guess which of these bonds will absorb at the highest frequency solely on the basis of the reduced masses. Solely on the basis of masses your answer must have been that the absorption fre quency falls along the series given. They rise because all along the series the electronegativity r . However. it should be easy to surmise that the vibrati onal frequency of a bond should increase with the strength of the bond. So we can safely say that C--C should have a frequency lower than that of C---C. N--H. double bonds are stronger than the single. The foregoing may make us think that we can predict the frequencies of vibration for a given bond. and F--H.. The actual observation is quite to the contrary. Also when the reduced m ass of the system decreases. One example will prove this. C--H . T he frequencies rise along the series.0xlO5gs-2 7 2x22 = 9. To an extent we can. g.ises and with it rises the force constant. NH2. Some of these bands are called overtone bands. So far we have been talking about vibrations which are dubbed fundamental.6. These bands are generated by modulation of fund amental . Also. Normally each vibration mode absorbs a t a different frequency.. there are vibrations whose absorption frequency may lie outside the normal infrared region examined. etc. methane should have 9 and ethane 18. 3 fundamental modes of vibrations. Thus.). Such vibrations are ca lled degenerate. Modes of Vibration The theory of molecular vibrations predicts that an asymmetrical molecule which contains n atoms will have 3n . Likewise. There are other frequencies at which bands appear in.23 shows vibrational modes available for AX systems (any atom joined to two other atoms.6 modes of fundamental vibrations. Thus a CH group may give rise to two C--H stretch bands. e. this is not always true. symmetric an d asymmetric. There will be some vibrations which may absorb at the same frequency. some caution . CH2 . NO2.must be exercised in doing so. Figure 8.e. However. although it is possible to predict the frequencies in a general manner tak ing the help of Hooke's law. This means that a mole cule like CO2 o should possess (3 x 3) . Naturally their absorption bands will overlap.an infrared absorption spectrum. i.. polysaccharides). substances that give-the same infrared spectra are identical. the strength of their bonds. -. Such interactions may take place either as x + y. a strong absorption at 1700 cm.may give rise to a weaker absorption at 340 0 cm-I.and 1400 cm-. The combination bands. You would agree that combination bands are unique to a compound. The molecules in which biochemists are more interested are the biological macrom olecules (proteins. this region is dubbed the fingerprint region. a particular kind of combination can only occur in that compound and in none other. Thus. They are going to have an effect on the exact frequency it absorbs. The vibrational frequencies therefore reflect the structur e and conformation of a molecule. Conversely. In summary we can say as under. strong absorption at 800 cm-I may give rise to a weaker absorp tion at 1600 cm-.. If this bond exists within a given molecule. Since these molecules have a very la . Since each comp ound has a particular arrangement of atoms. t he other atoms present in that molecule are bound to have an effect on the way this bond behave s. we cannot hope that this particular bond is in isolation from the res t of the molecule. T he resulting weaker absorptions are called beats. Thus if we refer to C-H stretch frequency. The infrared spectrum of any molecule is thus absolutely specific and is therefore known as t he 'fingerprint' of the compound. Any given bond in a molecule does not exist in isolation. We may therefore say that the frequencies of molecular vibrations do n ot depend just on the masses of the atoms concerned and the strength of their bonds.y. Since the atoms involved and therefore their masses. the other bonds in that molecule. or as x . Thus a C--H bond in o ne compound may vibrate at a slightly different frequency when compared to a C--H bond in a different molecule. x and y. assume extreme imp ortance because they may be the signature or the fingerprint of a given compound. A larg e number of these combination bands fall in the region between 900 cm. therefore. For thi s reason. nucleic acids. and the arrangement of atoms differ from compound to compound (u nless the compounds are enantiomers). i nteract with each other (combinations).it may be said that the infrared spectra of no two co mpounds are alike.212 Biophysical Chemistry vibrations. Another kind of modulation is when two different frequencies. but also o n the arrangement of atoms. 24. it will have (3 x 100) . And yet the infrared spectra of even thes e compounds will provide information about the various functional groups which absorb at characte ristic infrared regions regardless of whether they are present in a large or a small molecule ( later we will see that even conformational studies on macromolecules are done with the help of inf rared spectra).rge number of atoms. The importance of infrared spectroscopy to the study of the functional groups ca nnot therefore be overemphasized. Conside r a molecule which has 100 atoms.6 = 294 fundamental modes of vibra tion not to say anything about the bands that will appear out of combinations and other modulati ons. a substance often used for taking infrared spectra of solids. that a rigorous explanation o f their infrared spectra will be unattainable. . they will have an enormous number of possible modes of vibration. Biological macromolecules have much more than 100 atoms. Infrared Spectra of Common Functional Groups The regions in which functional groups absorb are summarized in Figure 8.ubiquitous occurrence. The figure also illustrates a very simple spectrum belonging to the liquid paraffin Nujol. It will be apt for us to spend some time discussing the vibra tions of dlfferent functional groups of. These macromolecules will possess such an enormous number of fundamental modes of vibration. 24. 1 C "--O 1 | bending and corn-| N-----oCN |bination bands. IR spectrum of the Iklukl hydrcx :oybon.1028 213 tching I Spectrophotometry 4000 3500 3000 2500 2000 1500 1000 cra'* N--H hing JL c--c I r Other stretching. is olso . | Stetc I'll | The fingerprint | Bending [ region j Figure 8. A summary of the regions of absorptlon correspondgng to the stretch ing and bending vibrations of the ma functlonal groups and carbon skeleton bonds. NuJol. The greater the steength of the bond between two similar atoms. and carbon and nitrogen absorb just below the region where single bond stretchin g involving hydrogen absorb. However. N. The Carbon Skeleton Aromatics: C--H str. N--H bending vibrations also absorb in this region. C--H def and a group of overtone combination bon ds are seen in infrared spectra of aromatics. In a book of this size and generality. 2. it is not possible to discuss the vibrati ons assignable to each and every functional group. C=-'C str.seen. carbon-nitrogen.may not be seen frequently. three. Obvious from the figure are some rules that must be borne in mind. Most aromatic compounds show three of the four possible OC str bands. 3. a cursory discussion of major funct ional groups in structures that may be encountered frequently-in biochemistry follows. and the two bands around 1600 cm-I may coalesce. Thus.--H bending is an exception to the rule. stretching vibrations of triple bonds between carbon ato ms. Of these. the band at 1450 cm. Thus these bands are characteristic of the substitution patte rn on the ring. the stretching vibrationsf single bonds involvi ng hydrogen give rise to absorption at the high frequency end of the spectrum. The p-substituted benzenes present a strong band above 800 cm . and nitrogen-oxygen absorb. 1. Bending vibrations are of much lower frequency and usually appear in the flngerpr/nt region below 1500 cm-'. carbon-oxygen. The number of hydrogen atoms on the ring may be characterized by out'of-plane C--H deformations. Thus. two. or four bands may be expected for C'-C sir. Owing to their low mass. the high er the frequency of the vibration. C--H sir occurs Just above 3000 cm-. Thes e bands may be quite weak. Below this is the region where stretching vibrations of double bonds bet ween carboncarbon. 4. In saturated esters the C---O stretch band occurs around 1740 cm-. it is all but impossible to det ect it. characteristic feature of alkenes may be the strong overtone bands for out-ofplane C--H def vibrations. In enols theCO stretch frequency occurs very low at 1580 cm-. Out of these . the band with the higher frequency is the one assignable to the antisymmetric stretch.2000 c m-. Cas4nyl Group The stretching absorption of this group has probably been studied more than any other group.214 Bophyscal Chemistry Alkanes and Allcyl Groups: Most commonly C--H str absorptions appear Just below 3000 cm-. the former are easily detectab le. If the carbon . In aldehydes. Alkynes: Out of terminal and nonterminal alkynes. For the alkyl groups the antisymmetric CH3 def occurs aro und 1390 cm-. Just below 1800 and Just above 1800 cm-. C-C---O (kete nes} absorb at 2150 crn-.two. The weaker CC sir band occurs near 22 00 cm-i. and --N3 (azides| absorb at 2140 cm-. The nonterminal alkynes have an extremely weak C-=C str and they lack the C--H s r absorption. where few other absorption appear. --N---C-S (isoth iocyanates} absorb at 2100 cm-!. A strong C--H str band occurs near 3300 cm-. The most. Alkenes: These skeletons give absorptions quite singular to that of aromatics an d as such if an alkene occurs within an aromatic molecule. The combination vibration occurs around 1800 . two C--O stretch bands occur. C--C-C (allenes) absorb around 1950 cm-I. O-CO absorb at 2350 cm-. Cumulative double bonds give rise to strong absorptions around 2000 cm-. The CO stretch band is always strong and so the overtone is almost always visible around 3400 cm-. --N--C-O (isocyanates) absorb at 2250 cm-. The latter is for antisymmetric stretch. Thus. Trans-alkenes and cis-alkenes are easily detectable from each other: the former gives a C--H def band around 970 cm-] while the latter gives i t around 700 cm-. the band occurs lower around 1720 cm-. For tertiary alcohols the former occurs between 1300 and 1410 cm.|O--H stretch). If the CO stretch bands occur around 1700 cm. the possibility is that we are seeing a carboxylic group. and the O--H defis between I000 and 1100 cm-. Chelation with a nearby OH group weakens the C--O bond even further an d consequently the band is lowered even more around 1880 cm-.while the latt er also is sh/fted towards higher frequencies and occurs between I I00 to 1200 c m-. For secondary alcohols the C--) sir is situated more or less as for primary alco hols. For the carbohydrates the detection is made simple by the presence of an extreme ly intense OH sir centred on 3300 cm. Hydroxy Compounds For the primary alcohols C--O sir band is centred around 1300 cm-. but the O--H def is centred around I I00 cm-].along with a broad band around 300 0 cm. .and a C---O str ¢entred at around 980 cm-. C-O stretch bands for carboxylic acid anhydrides occur near 1800 cm:.yl group is in conjugation with a double bond or an aromatic ring. Now the two beams are reflected to a chopper which is rotating at a speed of 10 otations per second. NmH def occur s around 1600 cm-. This instrument optically balances out the differential between the two beams. The amplifier is designed to amplify only the alternating current. both the beams will have equal inte nsities and the current flowing from detector to amplifier will be direct. One beam is made to pass through the sample while the other is allowe d to behave as the reference beam.. The amplified signal is record ed by a penrecorder. For nitro compounds two NcD str bands occur. INFRARED SPECTROPHOTOMI'ER : MODE OF OPERATION The spectrometer consists of a source of infrared light. It is the function of the detector to convert infra red thermal energy to electrical energy. albeit slowly. In dilute solutions free NmH sir can be seen near 3500 cm-. and the other ranging from 1300 to 1400 cm-. The function of such a double beam operation is to measure t he difference in intensities between the two beams at each wavelength.and for isonitriles this ba nd occurs.Spectrophotometry 215 Nitrogen Compounds Hydrogen bonding in amines modifies both symmetric and antisymmetric N--H str bands. not alternating. The grating also rotates. T herefore . one ranging from 1500 to 1600 cm-. at the frequencies where the sample doesn't absorb.. just below 2200 cm-. The chopper makes the reference and the sample beam to fall on the m onochromator grating alternately. For nitriles the C eN. This rotation send s individual frequencies to the detector. or alternating current to flow from the detector to the ampl ifier.str occurs just above 2200 cm. Light from the source is split into two beams of equal intensity. emitting radiation thro ughout the whole frequency range of the instrument. On the other hand. This will l ead to a pulsating. the detector will receive a wea k beam from the sample while the reference beam will retain full intensity. At the wavelength where the sample has absorbed. kept dry and they m ust be handied by the edges only. The gas cell has NaCl windows a t the ends (NaCI is transparent to infrared). The flats must be. a liquid . Vapor phase : The vapor or gas is introduced into gas ce//s which are either abo ut I0 cm long or shorter with a series of internal mirrors (multi-pass cells} which refle ct the infrared beam back and forth lengthening the path-length. The flats must be kept cle.this kind of instrument is called optical null recording spectrometer. . More soph isticated instruments are called ratio-recording instruments. L/quids : Liquids are usually observed as a thin film between two infrared-trans parent windows. In these three phases the intermolecular forces differ significantly and thus it is imperative. that the data obtained be specified for the sampling technique used. or a solid. Samplin Technique The sampling techniques depend on whether the sample is in vapor phase.an by washing them in toluene or chloroform and they must be optically polished using jewellers' rouge. This is done by squeezing a drop of liquid between NaCl flats. In these instruments the int ensities of both sample and reference beams are measured and ratioed. Most organic compounds have too low a vapor pressure for this phase to be useful . etc. Polymers and fatty materials often g ive excellent results as solid films. however. Kaydol. Solids: Solid spectra are recorded in three different ways. mu//s. Pressure is appl ied to the flats to achieve the desired thickness. Once the mull has been prepared. as before. the sample may be ground with p ure KBr (0.due to OH group. It must be kept in mind that intermolecular forces are different in solids as co mpared to solutions.1 to 2. slight moisture does get in and that results in a band at 3450 cm. With all these precautions. The particle size should be lower than the infrared wavelengt h. less than 2 turn.e.01 to 0.).1 mm. it is squeezed between flat plates of NaCI and mounted for spectroscopy. The KBr must be dry and the grinding must be conducted under infrared lamp to not to allow the moisture to condense. However. or chlorofluorocarbon oils are used. if the sampl e contains water.0 per cent sample by weight). and deposited Mulls are prepared by grinding about 1 mg of a solid in a small agate mortar wit h a liquid hydrocarbon (Nujol. the sample is dissolved in a volatile solvent and then the solu tion is allowed to evaporate drop by drop on an NaCI plate. One disadvantage with Nujol is that it gives C--H str and C--H def bands. NaCI flats are used almost throughout the infrared region. Poorly ground mixtures tend to scatter more light and lead to erron eously low transmissions. For solid films. Thus if C--H vibrations are to be studied. Once the gri nding is done the sample is squeezed. To avoid bands due to mulling agents altogether. In such cases CaF2 flats are used. i. KBr d /sks. For these purposes hexachlorobutadiene. Nujol is transparent to infrared over a very wide range and for this reason Nujol mulls are very widely used. Mechanical ball mills are available for the purpose. Solid films are also used for infrared dichroism study. Nujol ma y not be a good choice. between optical flats. This is particularly true of those functional groups which take part . they become useless..216 Biophysical Chemistry The thickness of the liquid film varies between 0. Another precaution that should be taken with this meth od is that the grinding must be done well. studying the conformation of molecules. This is because water absorbs infrared powe rfully owing to its high absorption coefficient and a high concentration (55 M). This solution is then introduced into a solution cell made of NaCI. So/ut/ons : The principal complication here for biochemists is that aqueous solu tions cannot be used for infrared spectra. t he pathlength of the reference cell should be 95 per cent of the pathlength of the sample cell if the solution concentration is 5 per cent.H20 mixture. Applications of Infrared Spectroscopy Mainly four types of problems have been addressed by the life scientists employi ng infrared spectroscopy. The pathlength of the cell varies between 0. These are : identification of compounds. Thus solid state spectra are different from solution spectra. It is better to measure such substances in solution. For true compensation.in hydrogen bonding. These different crystalline forms lead to differences in infrared sp ectra. the number of resolved lines are far greater in solid state spectra. it suffers from the disadvantage of inducing severe conformational changes in macromolecules. Many organic compounds ex ist as polymorphic variations. However. and understanding interactions between molecules.0 mm. The sample is dissolved in an appropriate solvent to give a 1-5 per cent solutio n. However. This solvent dissolves quite a few polar molecules. This problem is addressed by dissolving the sample in DO or D20 -.1 to 1. A second cell containing pure solvent is placed in the re ference beam to cancel out absorption due to the solvent only. Another alternative is to use chloroform. however. since all polymorphism is lost in that stat e. assaying the rate of reac tions. it should be 96 per cent if the solution concentrat ion is 4 per cent and so on. . See ere c o protons for each cn atom.]ation of : Macromoleees ssess a lge number of atoms d as have numerous fde n braons. eflc acflons volg ese funcon oups eider ese oups e consum or generated e eaflc reacon c be assayed e help of red specoscopy. e rate ofe rcflon c meased by measg e rate of appece ofe cbonyl setcg bmflon. enfls we sply ide n ese oups e help of ed scoscopy. d su atoms exchge eff protons e protons of ter. when e scte lete completely it was fod to be CHa (CHz)3CH=CH(CHz)7CHzOH. e protons to 0gen. e comunds e idenc. Sups we susct at a ven comund is idcnc to a o ple.trophotometry 217 Ider. oaon about vous ncn oups of uo compound is obted nog e ons wch e abon bds appe. c compound posses ses elthcr one or one double nd. entuy. If it a hyl oup. My cflon oups bioloc (cro)molecules exchge eff protons e protons of water . We c erefore role out t re ven to e elemen fo. oer approach to idencaflon cs rccose to idencafion of funcon oups.+ HDO H+DzO D+HDO What is happening in the above reactions is that an atom of mass number 1 is bei ng for an atom of mass number 2. ff e subsate possesses a hydrol oup d e product doesn't. If a double bond volg cn Is present. e loss or appellee of or en sg of bds has been of oer benefit to e bihet. s seeg disadvge c be actuy adve c nse at tt pruces a deed red spec which secs as c et of e concerned mole. us. e mpound sho ese bds d us crc Is a double bond n d e lone ogen Is pnt as a hyo l oup. Consider a substance which has the elemental foula CHO. e hyl oup d e cbon-cbon double bond as predicted by e red data. we have to do is to te r sa ofbo csc compounds under flenfl confions . ogen. e ae sd protons be exche d for deuterons. e compound must show a sh bd beccn cm. e elcment foula shows at c md has a lone ogen atom wch maybe present as a cbonyl oup or as a hydrol or as cer. R ofr: Ied speca we wonderful dicaflon for my funcon oups. t us usatc s approach the help of example. us. NH + DzO ND + HDO @H + DzO D . e icd spectm must show a sh bd bceen 16 -18 cm-. Here. If e d speca match. From our discussion above we know that as the . it ssesses go. Or ff e subsate ds not possess a cnyl oup d e product does. If a prot is sIv DaO. when a molecule le prote is dissolved water. e rate of e cflon med by measg e rate of sappece of e H setcg bmflon. A shift tells us about the groups that exchange slowly and a fast shift tells us a bout. .reduced mass increases the frequency of absorption should decrease. Thus the ban ds of for the groups which have exchanged their protons shift to a lower wavenumber.for COND--. the rate of exchange can be studied by obse -rving the am ide II band. For the peptide group.for --CONH-.and at 1450 cm. located at !550 cm. which exchange faster. amide I. it gives us information about the kinetics and therm odynamics of the conformational transitions of the proteins concerned. And yet. These bands are v ery sensitive to hydrogen bonding and have been extensively utilized for hydrogen bonding in biol ogical macromolecules and pertJnent model compounds. this is a first order r eaction with a half life of about 0. Table 8. The reason for this is not far to seek. The generally accepted nomenclature.7 Characteristic Infrared Bands of the Peptide Linkage Designation App frequency(cm-} Description A . The amide A. and the approx imate description of each mode is given in Table 8.218 Biophysical Cherntstry Why should these exchanges be of interest to us? They are of interest because th ey tell us much more about the protein than just the exchange.0 minute. In oriented samples these bands sh ow marked dichroism and can be utilized to study the direction of N--H bonds. This surely seems like a disadvantage as far as studying conformatio n of proteins with the help of infrared spectroscopy is concerned. infrared spectrosc opy is of considerable use in studies of protein conformation.1 to. the approximate frequencies. and amide II and amide V have bee n most frequently used for such investigations.7. in comparison. This would mean that their infrared spectra will contain a huge number of vibrations. The chemical polypeptide repeat unit gives rise to nine characteristic infrared abso rption bands. The amide A and B bands arise from the NH stretching vibration in resonance with the first overtone of amide II (essentially NH bending) vibration. 1. Study ofconformatlon : Proteins (and for that matter. any macromolecule) contain a huge number of atoms. Consider this completely den atured proteins and small peptides exchange their protons fast. Thus if we study the kinetics of such exchanges. proteins in their native state exchange very slowly with half lives extending into hours or days for complete exchange. Some bands are more useful for c onformation studies than the others. when it is part ofa [-structure. The reason for this importance is that the frequency o f this band depends upon the environment of the bond. and ff the bond happens to be present in a random coil. In addition. and at 650 cm. NwH bending NH bending OCN bending out of plane NH bending out of place C-----O bending skeletal torsion. the band appears at 1650 cm-. Thus. A nd if a protein contains all the three structures. when the given bond is a part of the a-helix. 700 for beta structure.B I II III IV V VI VII -33O0 } -3100 } 1600-1690 1480-1575 1229-1301 625-767 640°600 537-606 -200 NH stretching in resonance with NH bending C-O tretching C--N stretching. the band appears at 1632 and 1685 cm:. These bands are all well resolved. the band appea rs at 1658 cm-. the amide V band appears at 600 for alpha-helix. In such a case. all three bands will appear.for the unordered structure. The amide I band is of great value as far as the study of secondary structure of a given polypeptide is concerned. measuring the . one can never be sure whether the same proportion of the configurations also occurs naturally in the a queous medium. will give an idea about the proportion of amino acid s which are in a given configuration. since D20 is the solvent which is used. .intensities of these bands. Of course. even optically inactive compounds will show linear dichroism. This stretching of the film the sample in the direction of the stretching. Let's try and understand this application without going into the mathematics of linear simple fact is that absorption by an oriented sample will be maximum when the is parallel to the transition dipole moment of that vibration is to be excited. Thu s rat tail tendons.Spectrophotometry 219 There is a totally different mariner in which infrared spectroscopy is applied t o study conformations. The phenomenon used in infrared dichroism is One can refer to this as linear dichroism. have been studied. This phenomenon means that in one direction will absorb plane polarized light differently when the electric of the light is parallel to the orientation of the molecule and when it is perpe ndicular to Whether the compound is optically active or not is of no consequence to this . Such studies can also be done on liquids solutions provided the liquid is placed in the space between the two cylinders a nd the rotated. researchers make use of what is known as infrared In the next chapter we will be discussing the phenomenon called circular dichrol sm. Absorption will be zero when the dipole moment of the vibratio n is to the electric vector. This can be the sample is prepared in the form of a thin film. porcupine quills. there will be molecules all degree of angles with the electric vector. dichroism means the difference in the absorption of right. These two frequencies are for C--O and N--H stretching respec . If molecules in a sample are randomly oriented. Also. for every molecule which is para llel to the a molecule which is perpendicular to it. The conformation of fibrous proteins has been studied by infrared dichroism. The molecules then orient themselves with their long axes in the direct ion The importance of infrared dichroism studies can bdst be demonstrated with the h elp of following examples. Here. silk fibre etc. In these proteins the absorptions at 1640 cm-I and 3300 cm-I are maximum when the electric vector of infrared light i s perpendicular to the fibre axis. In such a situation there will no t be any Thus the trick of studying infrared dichroism is to orient the sample.and left-handed circu larly by an optically active compound. This film then can be attache d to drums which are gently rotated stretching the film slightly. This observation can only be interpreted one way .these two bonds in the peptide bac kbone must be oriented perpendicular to the fibre axis (Figure 8.25)./ N--H R---CH C O H N/ --T Fibre axis -. What is true for these two groups is true for the bases in the DNA structure too .--R of the po/ypept/de dm/n 0t st/k (otherflbrous prote/ns may have s/m//ar arrangem ent). o--c. Note than the C-O and N--H groups of the peptide backbone lie perpendicular to t he fibre axis. The bands 1600 and 1750 cm{stretching vibrations for aromatic rings) show greater intensities . This inference is backed up by other studies on fibrous protein structure as well.tively. In the Hooke's equation. Thus.l igand binding. This shift gives ev idence of the presence of hydrogen bonds and is studied by infrared spectroscopy. Th e presence of hydrogen bond here lowers the force constant of both these bonds to stretching. as we have said earlie r. Disadvantages of Infrared Spectrophotometry With all the numerous applications listed above. or even CCI4. the force constant is a ltered. other molecular interactions such as the protein.the first one of them specifically for the biochemist s. . This again automatically means that the bases are arranged perpendicular to the axis of the fibre. As an example let's consider the hydrogen bond between an NH and a C-O group. the-bands due to the vibrations of these functional groups also shift. or to put it in more technical terms. enzyme-coenzyme interactions etc. D20--H20 mixtures. Yet. infrared spectrophotometry suff ers from two major disadvantages -. The problem is that the data ob tained about any molecule or situation in such solvents may not necessarily be true for aqueous solutions. The reason that hydrogen bonds can be studied with the help of infrared spectros copy is that the strength of the bonds of functional groups involved in hydrogen bonding is altered. Once the strength. What about the bending vibrations? Hydrogen bonding places restrictions on the b ending of these bonds. force constant occurs in the numerator. The best solvent for many biological works is water. water absorbs intensely in the infrared.in frared spectroscopy.220 Biophysical Chemistry when the electric vector of infrared light is perpendicular to the axis of DNA f ibre. Apart from hydrogen bonding. the bands attributable to bending will occur at hi gher frequencies. It is obvious that the frequency of these stretching vibrations will be reduced. are also studied by infrared spectros copy. Consequently. So do the two stands of DNA. Iateration between molecules : Polypeptide chains form interchain hydrogen bonds . This is borne out by other studies as well is predicted by the Watson-Crick structure. chloroform. Hydrogen bonds have been studied very profitably using. biologists are forced to use other solv ents such as the DO. RAMAN SPECTROPHOTOMETRY When incident light is scattered by intervening sample molecules. the frequency of light scattered is the same as that of the frequency of the incident photon. Beer's law may not be obeyed. It has been of immense use in locating various functional groups or chemical bonds in molecules and also for quantitative analysis of complex mixtrares. the problem is of accurately measuring the pathlength of such a solid sample. This phenomenon is known as the Raman effect. This is again a problem related to the solvent. a small fraction of the scattered light is observed to have a different frequency from that of th e incident photon. Since its discovery in 1928 by Sir C. Such osci llations are said to be elastic.It is very difficult to obtain quantitative data with infrared spectrophotometry . First of all this method requires very p ainstaking and fine grinding because large crystal size would give rise to scattering. However.V. sc ientists use very small pathlengths. Even if this is done and the solid is prepared. This too is not desirable as far as quantitative results are concerned because short pathlength would require higher solute concentration. In order to minimize absorption due to any solvent that is used. Only those vibrations w hich cause a . For this the sample may be ground with KBr. At high concentratio n. during scattering of monochromatic light by molecules. Raman. There isn't possibly a solvent that is c ompletely transparent to infrared. the Raman effect has been an indispensable tool for the elucidation of molecular structure. An alternative is to do away with the solvent and to read the sample in the soli d state. such shifted lines. and (///) high light gathering power. radiation due to it i s cosiderably intense than the radiation due to Raman-shifted components. Those vibrations which do not cause a change in dipole moment and are consequently ina ctive in the are observed in Raman spectrn Thus. the m olecule absorbed the radiation while it was in the lowest vibrational state does not ret urn to the level. the emitted or the scattered will be of a lower frequency as compared to the incident radiation. There can be other present in the Raman spectrum. but to a level which is slightly higher. a charge displacement. If now. The primary function of a spectrometer is to reject totally radiation due to Rayleigh scattering and to de tect the Raman-shifted components. Le. The Raman spectrum. A collision between a molecule and a (in case of light scattering) does not lift the molecule to any quantized level. the two techniques provide complementary informations about of molecular vibrations and therefore about the structure and conformation of a Rayleigh scattering results when the . thus. Some molecu les (a less than 1 are capable ofindulglng in energy exchange before scattering These molecules give rise to Raman effect.26). These when some molecules absorb incident radiation when they are in a lower state. These lines are known as anti-Stokes lines. anti-Stokes lines may not usually be considered for chemical analysis. Th ese may subsequently decay to their ground state (which is lower than the state at w hich absorbed radiation) and consequently emit radiation which has a higher frequency than .sample molecules scatter light which is of the same as that of the incident photon. Since most of the molecules indulge in lyleigh scattering. provides detailed information about the vibrational spectrum of a given molecules. The Raman spectrometer should have (i) an intense light source. although the origins of Raman and infrared are quite different.Spectrophotometry 221 change in dipole moment. A laser unit serves as a good light source and the detection is usually performed by SPECTROFLUORIMETRY .. known as Stokes lines are observed in the Raman spectrum and to a different vibration in the molecule. Naturally we should infer that collision of the photon such molecules in not resulting in any energy exchange between them. rather the molecule can be thought to be virtually excited (see Figure 8. (t/) sensitive detection. are observed in the infrare d region. The differen ce in equal to the natural vibration frequency of the molecule's ground electronic sta te. 26). emitting quanta of radiation corresponding to each energy . Therefore. Since each quantum will have a smaller amount of energy. When an atom or molecule absorbs light. . the radiation emitted will have the original exciting radiation. The excited electron can now return to the ground state in eithe r of the two It might do so in one single step in which case it will emit light of the same w avelength Fhe electron might. the energy of photon absorbed lifts an e lectron higher orbital. after absorbing radiations.The phenomenon whereby a molecule. This shift toward a longer wavelength is known Stoke' s shift [Figure 8. emits radiation o f a as fluorescence. however. return to the ground state in a step-wise manner energy levels. fluorescence spectra are band spe ctra. Thus a compound which absorbs in the ultraviolet range. It is obvious that the emitted light will T different wavelengths corresponding to each intermediate energy level the elec tron on its Journey to the ground state. ' are usually independent of the wavelength of the radiation absorbed. Structural Factors Which Give Rise to Fluorescence Aromatic molecules or the molecules having multiple conjugated double bonds with a high degree of resonance stability generally fluoresce..seconds to Occur. --F. --I. OH.26Diagrammatic representation of Rayleigh and Raman scattering. --NHCH. --NO2..] enhance the fluorescence. Examples are amines with a lone pair on their nitrogen atom.222 Biophysical Chemistry First excited electronic state Virtual excited state {Laser frequency) . . Fluorescence is often given by molecules containing a nonbonding pair of valence electrons. Note th at no energy exchange takes place in Raman scattering (B and C).--Br. etc.. " I 'l -[ .. The higher the number of n-electrons.) decreas e fluorescence. hCOOH. Substituents strongly affect fluoresc ence...3) and therefore can provide information about events which take less than 1 0.. ' ... see Bo x 8.seconds or less. etc. On the other hand substituents which delocalize the n-electron [-NH. . Thus polycyclic aromatic compounds are more fluorescent than benzene derivatives. N(CH)2.. the higher is the fluorescence. OCH. (A) (B) 1 Excited vibrational energy levels Figure 8. Fluorescence is an extremely short lived phenomenon (10. Electron withdrawing substituents (hCl.. --NHCOCH. Both classes of substanc es possess delocalized n-electrons which can be placed in low-lying excitec single states. Intensity and wavelength of fluorescence of the same compound may vary with the solvent used. Solvents exhibiting strong van der Waal's binding forces and solvents poss essing electron withdrawing groups diminish fluorescence. . especially those changes which affect the charge status o f the chromophore influence fluorescence. glassy state and high viscosity are all pr omoters of fluorescence. Thus aniline fluoresces at neutral and alkal ine pH but becomes non-fluorescent at acidic pH. rigid and sterically uncrowded are the most fluoresc ent.Molecular rigidity is conducive to fluorescence. Aniso!e which fluoresces at neutral pH is non-fluorescent at alkaline pHs. Thus among the aromatic compoun ds. Chelation of aromatic compounds with metal long often promotes rigidity and reduces intern al vibrations. Chelation thus promotes fluores. those that are most planar. " Drastic changes in pH. Low temperature. cence (this principle is exploited in measuring the concentration of many metal long). Beer-Lambert law. .ISMPL) where k is the proportionality constant (< I). Thus. is also applied to fluorometry in the following form log ISOL'ElCr -. The values of intensities of the incident radiant ener gy and the transmitted energy are indicated by ISOLVEr and ISLE respectively.ISAMPLE. The intensity of fluorescence is given by F = k (IsoLV. The intensity of radiation absorbed is therefore given by ISOLVET -. C is the concentration of the substance and b is the path length. which we have considered previously.Z/cr -.223 state Fluorometry : Theory and Instrumentation Fluorometry is an important analytical tool for the det6rmination of extremely s mall concentrations of substances which exhibit fluorescence.Ef Cb ISAMPLE where ef is the absorptivity of the fluorescent material. indicated in Figure 8. o ne monochromator is placed before the sample holder and one after it.10fcb) Rearranging F-Fn = 10fCb Fn Therefore. log = fCb For Cb < 0. (/) There are two monochromators instead of one as in a spectrophotometer.27 are : Sample Slit - .303 kIso.m.I0Cb} f k[SOLVENTIS written as Fn the relationship becomes F = Fn (I .Slit recorder Amplifier 224 F = kIso. F= 2.01. and (ii) As fluorescence is maximum between 25-30°C.VET (I -.. The instrumentation of a spectrofluorimeter differs from that of the spectrophot ometer in two important respects besides other minor variations.t eCb.p. The main components of a spectrofluorimeter. This equation holds good for concentrations as low as a few p. the sampleholder has a device to maintain the temperature. ----.Monochromator 2 Detector ...Gt Source Collimator or I . a monochromator usually a prism (P). Spectrofluorimetry vs. to choose the wavelength with which the sam ple is to be irradiated. (//0 a second monochromator (P2) which. Absorption Spectrophotometry Advantages (0 Spectrofluorimetry gives extremely accurate results when very low concen trations are used. Absorption Spectrophotometry. sensitivityof absorption spectrophotometry is taxed when I00 mg of serotonln is to be detected. absorption spectrophotometry is not at all acc urate. The fluorescent radiation emitted ly the sample is given off in all directions. one selects the activating wavelength while the other selects the fluorescent wavelength. At very low concentrations. spectrofluorimetry on the other hand can determi ne 100 pg of serotcnin with relative ease. and (v) an amplifier. but in most instruments the sample is viewed at right angles to the incident beam. Th is arrangement imparts great spectral selectivity to spectrofluorometers. Disadvantages (0 A drawback of spectrofluorimetry is a high degree of absorption of fluor . spectrofluorimeters can make do with quite simple electronics. To cite an example. enables the determination of the fluorescent spectrum of the sample. Speetrofluorimetry vs. ( The 'Stokes' shift enables use of two monochromators in spectrofluorimet ry. (/v) a detector. Thi s is generally not possible with absorption spectrophotometers. (///) Since there is a direct relationship between sample concentration and th e intensity of fluorescence. placed aider the sample. usually a photomultiplier suited for wavelengths greater than 5 00 nm.Spectrophotometry 225 a continuous source of radiant energy (mercury lamp or xenon arc). never be washed with a dete rgent solution. smoke carcinogens. This is known as quench/ng. mor e of spectrofluorimetry are given below. cho lesterol. However. some metal long). . filter paper and many laboratory tissues cause interference in fluorimetric assays because they can release strong fluorescing materials. Thus.escent radiation by the emitting sample itself. porphyrins. th iamine. drugs such as lysergic acid and barbiturates. serotonin and dopamine. 'organophosphorus pesticide s. The cuvettes used for fluorimetry should. The more common applications of spectrofluorimetry include qualitative analysis spectra and absorption spectra gives a fair idea about the identity a compound). Dissolved oxygen is a very effective quencher. (//) A note of caution : Detergents. The cuvettes may also not be dried by wiping them with tissue paper. such as cortisol. oestrogen. In old instru ments the problem of quenching is obviated by measuring a low concentration of the sample. if precise w ork is required. Ta ble lists fluorescence maxima of some biologically important compounds. and studies on protein structure (FAD containing proteins). Quenching also occurs due to impurities. The modem instruments use microcells. quantitative analysis (applications include assay of riboflavin. nitrogen is bubbled through the sample to remove oxygen. therefore. pH 7. pH 7.0 7.0 Water.0 Water.8 Characteristic fluorescence maxima of some compounds of biological int erest Compound Solvent Max Phenylalanine Phenol Tyrosine Tryptophan Riboflavin FAD Chlorophyll a Chlorophyll b Fluorescein Rhodamin B Indole BSA Ovalbumin Lysozyme Chymotrypsin Fibrinogen Insulin Water.0 7.0 pH pH pH pH pH 7.0 7. pH 282 303 303 buffer.0 Water.226 Biophysical Chemistry Table 8.1 N NaOH Ethanol Phosphate Phosphate Phosphate Phosphate Phosphate Water. i N NaOH 0. 7.0 Water.0 . buffer. pH 7. buffer.0 7. pH 7. buffer.0 Hexane Hexane 0.0 Water. pH 7. pH 7. buffer. Such compoinds afford important information about thi s interface. These probes are permeable to the plasma membrane and upon enteri ng the cytosol combine with calcium (chelation). Quin-2 AM. and Fura-2 are three fluorescent probes which allow us to assay intracellular fr ee calcium concentration. The probes have also yielded much information about the structural features of t . as pointed out earlier. vary with its mobility and also with the polarity of the environment. These studies assume importance because of the role of calcium in controlling cellular metabol ism. Studies with AN S have shown that structural changes occur in mitochondrial membrane during oxidative phospho rylation. This is especially so since the fluorescent properties of a molecule. (ii) Fluorescent probes and studies on membrane structure : Fluorescent probes s uch as anilinonaphthalene-8-sulphonate (ANS) and N-methyl-2-anilino-6-naphthalene sulph onate (MNS).348 520 520 665 650 518 630 325 340 332 340 330 332 303 (i) Intracellular free calcium concentration assay : Quin-2 (an EGTA derivative) . This chelation gives rise to fluoresce nce whose magnitude is directly proportional to the free Ca2÷ concentration in the cytosol. contain both charged and hydrophobic areas and therefore locate at the wa ter lipid interface of the membrane. (iv) Fluorescent microscopy : Spectrofluorimeter when combined with a microscope allows the determination of subcellular location of fluorescent compounds or of materia ls which bind fluorescent dyes. Thes e membrane potential changes can be monitored by using fluorescent probes such as Di-S-C3-(5).. and m erocyanine 540 (the latter is not so satisfactory).he plasma membrane. This technique has given very important information in the fie ld of immunology and pharmacology. presence of pathological immune complexes may be detecte d with the help of FITC conjugates. The presence of an antigen on the cell surface may be detected by . (No Assay of membrane potential : Membrane potential of excitable cells is regul ated within strict limits. Thus. Changes in this potential regulate ion entry into the ceils. It is being used to obtain definitive informatio n about the between pairs of loci in a macromolecular assembly. it is being exploited in genetic counselling specifically to determ ine whether the fetus is genetically normal. f luorescence changes under different conditions provide the necessary clues about the positio ns of amino acids residues in the protein. denaturation etc. 2. a carboxylic group.ven protein can be found out with the help of fluorescence studies. A few important empirical rules for interpreting fluorescence spectra of protein s are listed : i.. a great deal of information about the stru cture of . and This rule holds true in all cases except where the protein is known to contain components. (v) Since fluorescent emission is extremely sensitive tolocal environment. Such quenching can be due proximity to tryptophan. Usually it is the tryptophan fluorescence which is studied most often. ty rosine. The fluorescence of a protein is solely due to the amino acids typtophar. protein -protein interaction. Spectra and the StudF of Protein Structure As with absorption spectrophotometry. Since this technique alS° allows v isualization of chromosomes.Spectrophotometry 227 a fluorescence labelled antibody. Quenching also t akes if tyrosine is ionized. mostly due to quenching. This i s so because weak fluorescence. By using acridine orange. the composition of the active site. this technique allow s visualization of nucleic acids within subcellular organelles. Phenylalanine has the smallest of quantum potential of t . or an amino group. Here too. it ca n be used to mom'tor the kinetics and thermodynamics of the incorporation of a particular sub unit or substrate into a macromolecular assembly. Fluorescence due to whichever of the fluorescing amino is quenched. 4. cesium ion. ac rylamide) provides relevant information. nitrate. max of tryptophan to shorter wav elengths. Quenching of fluorescence by known quenchers (iodide. t he following may be considered : (a) The amino acid is internal. If a shift in . 5. that amino acid should be on the surface. (b) The amino acid is hidden in a crevice which is too small for the quencher to enter. Decrease in polarity of the solvent pushes . . (c) The surroundings of the amino acid might be highly'charged so that the q uencher is being repelled. one may conclude that the tryptophan is internal as well as existing in a nonenvironment. max occurs towards shorter wavelength when the protein has be en dissolved non-polar solvent.he three amino acids. 7. If a shift in . max. If a known quencher fails to quench the fluorescence of an amino acid. one may conclude that the tryptophan is on the surface of the protein unless the solvent is such that it has brought about large-scale conformational change in 6. max occurs towards shorter wavelength when the protein has be en dissolved solvent. 3. such a situation increases the intensity of the . extrinsic fluorescence. If the substrate quenches the fluorescence due to tryptophan in a given enzym e. . 1-dimethylaminonaphthalene sulfonate (DNS). Extrinsic Fluorescence There are macromolecules where no fluorescent group is present in the location w here it is desired. (/) It should have the ability to bind tightly at a unique location. 8. Even the type of charge may be determined. Such a method is given the name . and dansyl chloride. If positively charged quencher fails to quench. or quite near it. All the above named naphthalene sulfonates (ANS. the tryptophan might be present in the active site. Those preferred for use in protein st udies are ANS. To illustrate just how extrinsic fluorescence may be of help in studying maeromo lecules. (it) its fl uorescence should be susceptible to changes in the environmental conditions. rhodamine. the strength of their fluorescence increases while the florescence maxima shift to shorter wa velengths. These compounds are therefore. used to detect non-polar regions of a given protein. acriflavin and acridine orange. and (iti) the group shou ld itself not bring eonformational changes in the maeromoleeule in which it is introduced. To be used as a fluor. fluorescein. The most commonly used fluors for the studies on nucleic acids are ethidium brom ide.228 Biophysical Chemistry The last case may be verified by trying to quench through a neutral quencher lik e acrylamide. naturally. in such cases biochemists introduce a fluorescent group into the molecu le. If it is able to quench. the amino acid is in a positive environment. the chemical group must saUsfy cer tain requirements. DNS and TNS) have a peculiar pr operty of fluorescing quite weakly In the aqueous medium. Ditto with the negatively charged quenc her. proflavin. the existence of the amino acid in a charged environmen t is confirmed. 2-p-toluidylnaphthalene-6-sulfonate (TNS). The groups which are introduc ed in such a way are catled fluors. But in non-polar surroundings . wi th the help of acridine orange you may detect whether the sample contains single or double stra nded nucleic aeid. ANS has a property of giving no fluore scence when it is bound to haemoglobin. and If the sarple contains both. Haemoglobin molecule has two components -. the fluorescence vanishes and AN S is removed from binding. or single strands of DNA) it gives a red fluorescence.two examples have been cited below. Association with double stranded DNA causes it to fluoresce green. And that the site where the porphyrln binds to apohaemoglobin is highly non-pola r. But an association with apohaemoglobin causes it to fluoresce. I. . Fluorescence and Energy Transfer Given below are the excitation and the emission spectra of two different fluors. known as apohaemoglobin. Naturally. All t he more important is the fact that the fluorescent maxima are different for different nu cleic acids. x. That it fluoresces when bound to apohaemoglobin indicates that it is binding at a region which is . If the porphyrin prosthetic group is now added. 2. This indicates that ANS binds at the same site where the p orphyrin is.non-polar. Acridine orange has a property of an increase in fluorescent intensity as wel l as a shift in fluorescent maximum to shorter wavelengths when bound to nucleic acids. Bound to single stranded nucleic acids (RNA.the prosthetic group which is a po rphyrin and the protein. how much of each type is there. and y. excitation spec we me e rge of wavelens at a fluor must absorb to fluoresce. . (C) sn s w n sWn .oer raaflon as a mediator. e efficien of s press w later sho to 1 %.. e ession specm of x oveHaps e cition specm for y. In ese studies too it could demonsated at ener absorbed at ous ps of e prote mo lecules could ate to e locaon whe e acflvaon ced. e fluorescence due to y be obted. d y e e se soluon. ou lack of pointed discussion on e pot you e ven to at e excision specm is sonous e absoon specm for a fluor. ely as 1928 Wbg d Negele showed at t abrbed by e oflc o ads of a heme prote yeas t wch were poisoned by cbon monode pcipat sptg off e cbon monode radlc attached to e heme moie. ere were oer studies about acaflon of ees e help of t. wch en etted oer photon d alonger ve!en espong to its ession specm. was produced by absoflon of much smer wavelens. y fer een 1 2.to e. er oer ents so demonst e fact at abflon of t me comophores cod result fluor escence etted by oer comophores which we not cit dfly by e t. somees ngs turn out derenfly. Abson s d by s esn s d is. s is deed e case. In such a it may we we be at e luflon is adiated e of e cion of x.Spectrophotometry 229 I I Spectra for (A N. So. To be ct. I n such a case we c say at dion specm for y is derent from its usu abon specm. at happened? A loc reason for e phenomenon may be as foows: en x s excited. In s laon e ener absorbed by e t fluor is sfeed. Consider a system where go e fluors x. e . Note at e esston sc ofx oveHaps e excitaon spec ofy. it fluoresce. is detely is a possib but s is no t e reason for e ave phenomenon. x ab by y. N sn s of A os t son s ofB. Howler. f. as the distance between the molecules increa ses. 2. Three Conditions must. Since the experimental data fit the last mechanism. And this is exactly what makes the phenomenon of interest t o a biochemist. hydrogen radical.230 Biophysical Chemistry Several mechanisms were proposed for the transfer of energy in protein molecules . or (4) transfer by reson ance interaction between chromophores. be Satisfied for resonance energy transfer to occur. Thes e are 1. (3) transfer by proton. In fact the efficiency of energy t ransfer increases with the fluorescence quantum yield of the donor. Some of these were (1) transfer by electron migra. The energy donor must be fluorescent. 70/ seems to be the upper limit for efficient energy transfer. or hydride ion migration th rough an ordered array of water molecules surrounding the macromolecule. .tion in conductivity bands. The more they overlap. He postulat ed that the rate of transfer depends on the inverse sixth power of the distance between the donor and the acceptor. 3. In fact. the higher is the efficiency of transfer. Fluorescence energy-transfer is then some kind of a molecular ruler or a scale. The distance between the donor and the acceptor must not exceed 50-100 A . the efficiency of energy-transfer may decrease. Thispredicted distance dependence was verified later by experiment al studies of fluorescent donor-acceptor pairs separated by a known distance in defined system s. Let us See why. The absorption spectrum of the acceptor must overlap with the emission s pectrum of the donor. the transfer may ta ke place at a very high efficiency. If the two molecules engaging in energy-transfer are nearby. It is the last condition that makes this phenomenon of such large interest to a biochemist. this p henomenon has come to be known as resonance energy transfer. a biochemist can p redict the distance that may be there between two fluors lying within the same macromolecul e. (2) tra nsfer by exciton migration. We may then say that the eJficiency of transfer is a function of molecular distances. Because. In 1948 Forster proposed a theory for the resonance energy transfer. However. then by measuring the efficiency of energy-transfer. is the efficiency. Both th ese methods utilize three wavelengtths. . a nd R is a constant related to each donor-acceptor pair. and )a" The characteristics which th ese three wavelengths must possess are as follows : it must be absorbed by the donor effic iently but not by the aeeeptor: t2 should be a wavelength emitted by the donor but not by the a ecelStor. )h' 12.The mathematical equation that describes the eciency of transfer as a function o f molecular distance is as follows R6 where E. There are mainly two methods to measure E. R is the distance between the donor-acceptor pair. The above equation may be rewritten for R as follows = Ro(1-Ej R Let us see how E is measured. la should be a wavelength emitted by the aeceptor and not by the donor. The exci tation wavelength is From E one can measure R if To is known. the emission ate. Before energy-transfer experiments are performed. Let iv be the fluorescent intensity when the acceptor is absent and let fv. (2) extinction coefficie nt of the donor. A consideration of the orien tation factor is important owing to the fact that the fluorescence from the donor is polarized an d the acceptor may be at an angle with respect to the. The fraction of the donors that r emain excited is written as loE. To determine To one has to measure (1) the fluorescent intensity of the aceptor as a function of . while f is the fluorescence of acceptor at X when only the acceptor is present. moreover. However. Then The second method deals with measuring the fluorescent intensity of the acceptor at 3" The mathematical expression in this case is (^ V the fluorescence of the acceptor at 3 when the donor and acceptor both are prese nt. (2) There should only be one donor and one acceptor. There is another factor that must be considered but which is beyond the scope of this book. The first method excites the donor by using . This is the orientation factor. t he wavelength at which the donor emits. the following conditions must be met: (I) if a fluorescent group is to be attached. t he group itself should be inert so as to cause minimal perturbations in the macromolecular struc ture. and (3) the value of Q (photons emitted divided by the photons absorbed) of the donor when the acceptor is absent.Spectrophotometry 231 It stands to reason that if energy transfer is taking place. apart from the manner of attachment.^ be the intensity in the presence of the acceptor. the donor and acceptor groups are in rapid motion w ith respect to each other and the orientation factor is hardly ever higher than 1. both should be at kno wn chemical . plane of polarization such that maximum absorption may not take place.20. and then measures quenching at 2" Let f be the fluorescent intensity at 2 if the donor was excited using . it should be attached in a manner that it doesn't alter the structure of the macromolecule. should be quenched. This group is no good as an acceptor since it does not fluoresce. and pyr ldoxal phosphate are dlso attractive potential donors and acceptors. The heme group in heme proteins is a very good acceptor since it has an absorption spectrum covering the entire visible range. Once these condition s have been considered. Fluorescent coenzymes such as NAD÷. The first step in identifying a donor-acceptor pair is to ascertain whether the macmolecule has an intrinsic chromophore that can serve as a donor or an acceptor. the experiment can be carried out. FMN (oxidized). and (3) The To value for the donor-acceptor pair must be known and shou ld be in close agreement with the distance between the donor and acceptor.sites.. .. The galactose b inding protein from Salmonella possesses Just one tryptophan residue and this is wonder ful for energy transfer measurements. Tryptopha n residue in proteins serves as an excellent donor owing to the fact that its emission center ed at around 330 nm overlaps the absorption spectrum of many potential acceptors. and other adenine nucleotides Heme Thiamine diphosphate Fatty acids Phospholipids ethenoadenosine derivatives benzoadenosine derivatives protoporphyrin. CoA. derivatives of phosphatidyl ethanolamtne Th.Ca÷ 232 Biophysical Chemistry If a good donor or acceptor is not naturally orescent analogues are available. NAD÷. The available in the macromolecule. These analogues can be use d as labels or energy transfer experiments Biomolecule Fluorescent analogue ATP. Thus benzoadenoslne adenosine nucieotides.and -parinarlc acid phospholipids containing parinaric acid. Eu . FAD. flu derivatives are good analogues for tin derivatives are good analogues following table lists a few. Table 8. dialkyl cyanine dyes. Several fluorescent analogues are now available.9 Florescent analogues of a few biomolecules. zinc and tin porphyrin thiochrome diphosphate a. Protoporphyrin and its zinc and for heine. A few examples of the application of the energy-transfer technique to structure determination are given below. What is its s hape when it is in solution? Is it different or is it the same? Yang and Soll tackled this problem using energy-transfer studies. and (i) 3"-termlnal adenosine. Thus. (e) pseudouridine. and e from f by 55 amstrong units. d from fby more than 65. (see Figure 8. (d) 2-thiouridine at the anticodon. b from fby 38. c from e by 36. . The distances that were determined crystallographlcally are 25.29). except for the distance betwee n c and e where the two data do not agree well. and 53 amstrong units respectively. (c) dihydrouridine. or coumarin derivatives at two of the following sites : (a) the 5'-end. The co nclusion that Yang and Soll reached was that even in solutions tRNA adopts a cloverleaf struct ure. 41. Three-Dimensional Structure of tRNA In the crystalline state the tRNA exists in clover-leaf structure. dansyl . all other distances are very close.The distances that they calculated from observed transfer efflciencles were as follows: a was separated from F by 24.co/0 labeled with acridine. (b) 4-thlouridine. 23. 74. anthranilamlde. . They prepared five species of tRNA (either fmet or glutamate tRNA from E. pyntvate dehydrogenase complex -. positions at which j 'luorescent labels were introduced as well as the positions of unusual bases on the basis of which each arm is given a name. However. dihydrolipoyl transacetyl ase. The diagram shows the.Clover leaf structure of tRNA. CoA. Do we have any support for this hypothesis? The length of the lipoyllysine arm is known to be 14A. Pyruvate dehydrogenase complex is a multienzyme complex which converts pyruvate to It consists of three enzymes : pyruvate dehydrogenase. This can be more easily understood from the figure given below (Figure i. It has five coenzymes : NAD÷. energy -transfer have spurred proposition of alternative models. dihydrolipoyl dehydrogenase. 8. the energy transfer studies indicate that the distance between the a ctive sites of dehydrogenase and dihydrolipoyl dehydrogenase is at least 45A. "lipoic acid. thia mine and FAD. Now if the swinging arm h ypothesis the distance between the active sites of any two enzymes within the crystal must not 28].233 4. and (2) acyl transfer between ad jacent . The lack of evidence does not mean that the hypothesis is wrong. The enzyme dihydrolipoyl dehydrogenase is centrally placed in the a lipoic acid moiety attached to a lysine and it is hypothesized that it is this t moves around the crystal of the enzyme as a swinging arm bringing successive to the other enzymes in order that the reaction proceeds. However. Two of the main alternative mode ls are (I) substrate diffusion within the complex.29 . the arm c an reach them.greater than .CoA Dihydrolipoyl dehydrogenase {E3) Lipolysme arm O -< 28A o Dihydrolipoyl 14A transacetylase (E) Figure 8. (A) The lipoyllystne ann s 14 in length. Thus if the active sites o f the two other enzymes core placed a maximum of 28 A from each other.234 CoA HS CH3CO 0 14A {B} CoA Pyruvate dehydrogenase (E) Biophysical Chemistry CH3CO .30. (B) If the distance becomes. 28 . . the arm cannot reach the actlve sltes. It is the latt er which absorbs light. opsin and a fluor. both undergo a change in their three-dimensional stru cture. proposed the 'lock and key model.Ill +Ix p . E. In fact.Ill .. a large change in the distance between the donor and the acceptor gro ups takes place upon addition of the substrate. To check whether rhodopsin nergy-transfer experiments were performed. the change is such that they will fit snugly. This hypothesis can be tasted by energy-transfer studies. indeed. This means that rhodopsin cannot be spherical and. If the substrate is true. This is ample proof for the induced-fit mo del. 75.Ix Spectrophotometry 235 Changes in the Enzyme Conformation When Substrate Binds It was Emil Fischer who had. in order to explain the great specificity of an enz yme for a group of substrates. This fluor acted as the of this size wo is spherical. must possess a n elongated protein. The substrate behaves lik e a key. There are two parts to it: the protein. Rhodopsin Serves as a Light-Sensitive Gate Rhodopsin is the main light absorbing component in the discs of rod cells in the eye.' The model suggests that when a substrate approaches a given enzyme.. is a distance big enough to make this protein traverse th . e in cis-retinal. When such studie s are performed. Donor and acceptor gro ups are bound to an enzyme and the distance between them is measured when no substrate i s present. Then the substrate is added and the distance is measured again. Koshland improved up on this model when he suggested the 'induced-fit model. D." The model proposed that the active sites of the enzymes are like the key-space of a lock. The efficiency of transfer showed that the two fluors must be about 75A away fro m each other. A spherical molecule uld roughly have a diameter of about 45. cis-retinal. The molecular weight of rhodopsin is 40 KD. acceptor. The wrong substrate has the wrong shape and doesn't fit snugly. The correct substrate is like the correct key for a given lock which fits snugly int o it. The protein already has a good donor Another fluor was tied to a sulfhydryl group in opsin. And several lines of evidence suggest that rho dopsin indeed may be acting as a light-sensitive ion-flow gate. This intensity will be zero if the polarizer orientation is perpendicular to the plane of polarized light (Figure 8.31). the intensity o. A value of zero means that light is un polarized. Subsequent X-ray-scattering experiments confirmed the dimensions of rhodopsin to be just th ose found with energy transfer experiments. Ther efore. . The value for P varies between +1 and -1.f the transmitted polarized light will be maximum. for a partially polarized light. The above data encouraged researchers to hypothesize that rhodopsin may actually be serving as a light-sensitive gate controlling the ion flow across retinal disc m embrane. the polarization P. may be given by the relationship Where I and Ii are the intensities observed parallel and perpendicular to an arb itrary axis. FLUORESCENCE POLARIZATION If the orientation of the polarizer is parallel to the plane of polarization of light. Other values mean that the light is partially polarized.e retinal disc membrane. The factors leading to this los s of polarization are explained below. In order to do sol a polarizer is placed between the monochromator)and the sample. if the exciting light is polarized. the fluores cence is found to be only partially polarized or even unpolarized. Production of plane-polarized light. Fluorescence polarization can be determined using the instrument shown in Figure 8. By Varying the axis of the second polarizer. both 11] and I±c an be determined.236 Polm'lzer Excitation Light source le holder Direction of propagation Figure 8. Waves of all orientations fall on the polarizer.31. The axis 9f this polarizer can be varied from being parallel to the fi rst polarizer to being perpendicular. monochromator Polarizer Emitted light m Emisslon onochromator .32. which allows only those waves to pass whose electrical vector is parallel to the axis of the polar lzer. A second polarizer is placed between the sampl e and the detector. Thus P can be measured. Usually. The sample is ecited with polarized light. . the chromophores randomly oriented. Consider a molecule. ed light a fluor? Will it emit the in the same plane as it absorbed? Not necessarily. but by the transition dip ole The latter is not generally parallel to the former. This value of polarization is called the intrinsic polarizatio n. P be less than 1. In a very short time. What if the molecule. The motion we . In-a solution then where the molecules are randomly oriented. th is will be even more true. that is absorbing the plane polarz. there are other factors that affect polarization and therefore intrinsic polariz ation is the value that is observed experimentally. This motion will transition dipole orientation and will cause loss of polarization. Note the fact that the transition dipole moment is not generally parallel to the electric This is the major factor for the loss of polarization. It has absorbed polarized light.237 A chromophore absorbs polarized light maximally if the direction of the electric vector of is parallel to the electric dipole moment of the chromophore. The probability of emission of with the plane of polarization at an angle with respect to the transition dipole is proportional to sin2. it w ill be emitting But in the short time between absorption and fluorescence. This non-parallel relatio nship the two dipoles ensures that even if the absorbing molecules are perfectly align ed with of polarization of the light being absorbed. If thi s angle is O. there would still be loss of polari zation. is a result of th e fact that the are not parallel. its value. Po. Others w ill have electric dipole moment at various angles to the plane of polarized light. it moves. In a solution. This is so because the polari zation light is not determined by the electric dipole moment. Only a few of them will satisfy the above condition. the probability of absorption of polarized light is cos20. This loss of polarization ( Pvalue being less than 1) is called So far we have seen that fluorescence polarization. the less will be its rotational mobil ity. The second factor is that of energy transfer between identical molecules. . the efficiency of tran sfer " . As the distance increases. take place when the dipoles are not parallel.are about here is rotational motion and not translational motion because translation al is unable to change the orientation of the dipole. the more the energy transfer. . the will be the loss of polarization. less the distance between the molecules. the more will be the rotationa l motion. Althou gh resonance transfer takes place with highest probability between molecules having parallel dipoles. The higher the viscosity. The higher the temperature. The larger the molecule. the lower the rotational mo tion. the lesser be the loss of polarization. To explain with the help of an example . but also depends upon the viscosity of the solution . The more the concentration. as well !the temperature. naturally there is'a In the previous pages we have seen that resonance energy transfer is a of distance between molecules. The rotational motion of a molecule is a function only its own size and shape. the more the loss of But concentration here is a complex term. The other factor that affects polarizatio n r is the energy transfer that may occur between the molecules. Thus macromolecules show substantial polarization o wing to really decreased mobility. energy transfer is also affected by concentration. the polar ization may be very near that of the intrinsic polarization since the molecule will elic it very r rotational motion). Rotational motion is therefor e one of that affect fluorescence polarization. When this occurs. the be the extent of loss of polarization (in fact at very high viscosity. In fact both the above factors are complex. The higher the size of the molecule. the lower will be the rotational mo bility. On the other hand. 5. If these small fluors are attached to another molecule (which may itself be polarization will increase owing to the larger combined size and reduced mobility. polarization. attached fluor. 3. if the viscosity of solution is ve ry low.in these conditions it be due to energy transfer. loss of polarizition will take place . The major conclusion that derives from the above discussion is as rotational motion. In this case polarization is quite high.238 Biophysical let's conjure an image of a fluor bound to a macromolecule. " After having considered energy transfer as the second factor affecting arrive at a good relationship. Concentration in suc h refer to a low concentration of the macromolecule-fluor complex in the solution. polarization decreases. if the binding is such that the f . The higher this d istance. lower we will say the concentration is. 4. On the other hand. Even if we completely immobilize a molecule by in creasin of the solution to a very high level. A fluor bound to a macromolecule shows good polarization because its mob ility greatly reduced by such binding owing to the large size of the macromolecule*. Small molecules rotate freely and thus do not give any polarization (P value is zero) 2. the higher will be the polarization. the loss polarization will mainly result from rotational motion of the molecules. If the binding is rigid (intercalation between bases of DNA) the m obility ¢ the fluor effectively means the mobility of the whole molecule. refer to the distance between the fluors on the macromolecule. I. We need to understand the consequence s of this rule as well as understand truly what we mean by rotational motion. The nature of binding of the fluor to the larger molecule also affects t he extent. but for larger proteins. 7. the larger structure will have lesser rotational movement. to observe depolarlzation due to motion. What the intrinsic fluorescence of the macromolecules and its polarization? For examp le the fluorescence of tryptophan in a protein and utilization of its polarization in s tudying the There is a problem here. extrinsic fluorescence has to be ma de use of. We have consistently talked about the polarization of a fluor bound to a macromo lecule. it will result in reduced rotational mobility and higher polarization. the lifetime of the excited state should be suffic iently long. . 6. If the fluor is bound to such an associating system. polarization will increase with association. If the macromolecules undergo polymerization. T hus. there should be a good time lag between excitation and emission so that the molecule m ay movement in that time and depolarization may occur.e. On the contrary.luor move without the whole macromolecule moving ( binding to an amino acid side which can move with respect to the polypeptide chain) the polarization may not high as in the first case. i. if the conformational change leads to diso rder the rotational motion is slated to increase and the polarization will decrease. . Large proteins move very slowly on a molecular scale. int rinsic may be of some use. For very sma]l proteins. If the fluor is bound to a macromolecule which changes its conformation. its will be affected: If the change in conformation leads to the macromolecule assum ing more ordered structure. the resultin g is large.239 Given below a. The trick is in tagging dansyl to the polypeptides.. The effect of various environmental factors. Conditio ns flexibility of a protein will decrease polarization by giving rise to higher rot . and the the association kinetics has also been studied using the fluorescence technique. as as for the substrate enzyme interaction. Studies on Proteins The enzyme lactate dehydrogenase is a tetramer. such as the pH. Naturally. It is also seen that depolarization and loss of activity occur in hand meaning that the tetramer is the active form while the monomer is inacti ve. Tag an extrinsic fluor to an antigen. These systems too have been studied usi ng polarization. in Proteins If one can tag an extrinsic fluor to a protein without changing its conformation . then in protein conformation can be studied using fluorescence polarization. the ionic strength. This binding rise to the antigen-antibody complex which will be large. The association/dissociation pro perties been studied using fluorescence polarization. This can be used to detect the presence of specific antibodies in a given mixture. binding will occur.e a few examples of use of fluorescence polarization to biochemis try. No t just Even the kinetics of binding can be assayed with the help of polarization. What is true for the antigen-antibody interaction is true for receptor-ligand in teraction. Incubate the antigen with a mixture of ant ibodies. When the tetramer depolarization occurs. When the monomers associate to form tetramers. Thus polarization increases as association occurs. the polariz ation to the extrinsic fluor should increase owing to lesser rotational motion of the complex. If antibody specific for the antigen is present in the mixture. . disulfide linkages be present (2-mercaptoethanol destabilizes disulfide linkages and will thus incr ease the protein concerned). If treatment protein with 2-mercaptoethanol reduces its fluorescence polarization. The method may even be made quantitative for measurement linkages.ational of different proteins and the different conditions that contribute can be studied. Presence of disulfide linkages can also be studied with the help of this techniq ue. . so for spectrophotometers. luminol) with hydrogen peroxide in the presence of m etal which act as catalyst. and (iii ) a detector which usually a photomultiplier along with suitable amplifier and a recorder. The li reaction taking place in the cuvette is measured either as a peak value or as th e rate of chan in the intensity. Since luminescent light is virtually monochroma tic as result of its emission through a specific reaction. The second advantage concerns instrumentation of luminometers. The basic featm-es of luminometers are (i) a light tight chamber in which the containing the sample can be placed. acridinium salts and luminol. Although this limit is not achieved i n than femtomole quantities are measurable.g. As compared to chemiluminescence. Luminescence has been classified into two categories. commercial availability of luminometers.. are not required in luminometers. Luminescence produced by the intervention of an enzyme is known bioluminescence. elaborate wavelength selecto rs. Theoretically as lit tle as one (I0-8 mole) of the analyte may be assayed. . Applications The awareness of the advantages of luminometry have prompted improvement in. The phenomenon is usually ascribed to taking place in solution producing molecules in an excited state. Luminescence produced by chemical means is known as chemiluminescence. The chamber is also thermostatically contro lled. While the former method serves to measure the of interest. bioluminescent systems conver t and a higher proportion of energy as photons. First o f they are more sensitive as compared to spectrophotometry. Example can be cited of oxidation of luminescent compound (e. of luminescent compounds are luciferin.LUCIFERASE 240 Biophysical LUMINOIVITRY Luminescence may be described as the emission of light from a chemical the temperature of incandescence. Luminescent Measurements Luminescent measurements have several advantages over spectrophotometry. the latter is suited for measuring enzyme activities. facility for addition of luminescent reagents in a llght-tight fashion. While some release energy in the form of heat. some others release it in the form of photon s. Three systems in frequent use are firef ly. creatine phosphate and triglycerides.luminescence and luminol chemiluminescence. this method has been used to assay numerous ATP-specific enzym es and their substrates such as creatine kinase. Luciferin + ATP + 02 Oxyluciferin + AMP + CO2 + Photon (562 nm) The reaction stoichiometry is such that for each molecule of ATP reacting. The principles and applications of e ach systems are described below. is the most s ensitive method for ATP measurement and much less than one f mol (10-s tool) of ATP can b e measured. one p hoton of maximal intensity at 562 nm is prodiced. (i) Firefly luminescence and ATP measurement : Luciferase. By linkage to another reaction. The method. in the presence of ma gnesiur catalyzes the following reaction. therefore. The system becomes absolutely specific for ATP if all the reagents are purified. . Luminol may be oxidized at neutral pH if peroxidases or vario us other oxidases are used as catalysts.CHO + 02 OXIDOREDUCTASE ) NAD{P)" + FMNH2 FMN + R. (ili) Chemiluminescent assays using luminol : Several compounds have been used f or . O H H CO0+ 2HO + 20H[ "COONH2 O . The system utilizes a purified oxido reductase (specific either for NADH or NADPH) obtained from the bacterium Beneckea harveyL The sequence of the oxidoreductase reaction coupled to a bacterial luciferase is as follows NAD{P]H + H+ + FMN FMNH2 + R. The system can be utilized to measure numerous oxidoreductases which utilize NADH or NADPH. The method allows about 100 fmol of NADH to be assayed. The substrates of such enzymes can also be assayed by this system. chromium. iron or haemin compounds a re used as catalysts.hemiluminescent systems. Luminol is oxid ized by hydrogen peroxide at pH 10-11 if copper.Spectrophotornetry 241 (tl} Bacterial luminescence and measurement of coenzymes : The coenzymes that ca n be measured by this system are NADH and NADPH.COOH + H20+ Photon {495 nm) A photon of maximal intensity at 495 nm is produced in the reaction in which the bacterial luciferase catalyzes the oxidation of an aldehyde by oxygen in the pre sence of FMNH2. but luminol has been the most popular. a marine bacterium possesses lux genes which are involved in th e emission of light. Use of Genetic Engineering in Blosensors Various laboratories world over are trying to develop biosensors that are based on the ability of genetically engineered bacteria to emit visible light in response to specific compounds.NH2 + N2 + 4H20 + Photon {430 lu) Photons with maximal intensity at 430 nm are produced. The ease of measuring light makes this type of biosensor especially a ttractive. the photon efficiency is only 1% and the method is capable of assaying 1 pg of hydrogen per oxide or 0. Lux A and the lux B gene code for two subunits of a heterodimer flavin manoo-ygeis-e luciferase which catalyzes light production by oxidation of a reduced aldehyde. genes. and E genes. The following example will make the understanding of the above approach easier. luminol has been much used as a marker in luminescent immunoassays . Vibrio Jischeri. The lux operon consists of five structural and two regulator) . D. However. The system lends itself to measurement of hydrogen peroxide produced by specific enzyme methods from such substrates as glucose or urate. Aldehyde production is catalyzed by enzymes encoded by lux C. I I I I I I I I I I I I I I I I I I I I . Additionally.1 pg of peroxidase per ml solution. I I I I I I I I regulatory C D A B E . Serratia marcescens. Bringing the lux genes under the control of the promoters of t hese gene systems may give rise to biosensors for other elements as well. it is the absorption or emission of specific wavelengths by excited atoms that iS studied by this technique. possesses the mer genes. which are invol ved in mercury detoxification. genes for detoxification of other elements are also fo und in certain bacteria. but also the/uxgenes are induced owing to the mer promot er controlling the expression of both.242 Biophysical Chemistry Another bacterium. These E. The characteristic emission spectrum of the element is produced w . Naturally. Both the vari ations will be dealt with simultaneously. E. The general method of flame photometry can be applied in two complementary ways: emission flame photometry and atomic absorption spectrophotometry. New York. emitted light which was easily measurable. coli cells were transformed with this plasmid. The-optical system and even the photo-detectors used in spectrophotometry and fl ame spectrophotometry are identical. when placed in a medium containing mercury. Like the mer gene system. have recently fused these t wo operons into a plasrnld so that the lux genes are situated downstream of the mer genes a nd are therefore placed under the mer promoter. a/. whenever mercury is present in the med ium. Geiselhardt et. This is then an i deal biosensor to measure mercury in any given medium. not only are the mer genes. These genes are inducible genes and are transcribed only when mercury is present in the medium.colicells. Volatilization of molecules in a flame produces free atoms and then excites them to higher energy levels. of ClarksonUniversity. Conseq uently. FLAME SPECTROPHOTOMETRY The flame photometric analysis method is more or less similar to that of spectro photometry with the exception that the place of the sample cell is taken by a flame. sodium which gives a very high background emission is measured first an d the quantity of sodium determined is added to all the standards. standards for each of the components are prepared which cont ain the previously determined concentration of interfering components. This is known as cyclical analysis. the energy absorbed or emitted is proportional t o the number of atoms present in the optical path. Moreover. To relieve this deleterious effe ct certain other elements known as the releasing agents (strontium or lanthanum) must be added. The amount ofenergy emitted also depends upon the (t) temperature and (ii) compo sition of the flame. Atomic absorption spectrophotometry. measures the absorption of a be am of monochromatic light by atoms in a flame. such as the alkali m etals. This is the principle of emtssionflame photo metry. Certain elements. The need t o maintain flame composition constant also dictates which element should be determined firs t. some substances like aluminat e and silicate cause a decrease in emission of other elements. Thus. enhance the emission of other elements. It is therefore very necessary that the two flame variables must b e maintained constant and that standard solutions be used to calibrate the system. atomic spectra are abso lutely specific for the element involved. on the other hand.hen the excited atoms return to their ground state. After this. flame photometry also provides information about the quanti ty of the element(s] present. apart from giving the identity of th e element(s) present in a sample. On the other hand. Since the transitions available to the electrons in any given atom are specified by the available energy levels. Another way to deal with interferences is to measure all interfering components in a given sample. Thus. . e.. liq uid ashing. Nebulizer Air from -. is assayed immediately before and after the sample solution. Very frequently. if added. oxidative digestion of the sample in hydrogen peroxide/concentrated sulphuric ac id solution may be carried out.an cause large errors in flame photometric analysis. This meth od is known as bracketting. A small amount of selenium sulphate. The individual components are discussed below. internal standards are used and the choic e element for this purpose is usually lithium.. co Filter Sample Detector Recorder . while lithium sulphate is sometimes added to raise the boiling point. Calibration cues should be checked or reconstructed when the assays are carried out. This is so s ince even the good quality glass containers release metal long. a standard solution containing more or less the same concentration of the element as the sa mple solution.. Alternatively.. Flame instability c. therefore. the ashing is usually carried out under low temperatures in the presence of oxygen.Spectrophotometry 243 About two to three such cycles are necessary before an accurate idea of the quan tities of all sample components can be deermed Organic material in biological samples (which might cause interference) is usual ly removed by ashing.33. If high accuracy is desired. To prevent more volatile elements from getting sublimated. i.. It is. Instrumentation for Emission Flame Photometry The basic components of a flame emission spectrophotometer are shown in Figure 8. essential that all assays be carried out in triplicate. acts as a cat alyst. It is advisable to store samples md standards in polythene bottles. the nebulizer becomes the most critical part of a flame photomet er so much so that the efficiency of an analysis depends on the efficiency of the nebulizer. This leads to a considerable drop in pressure leading to a suction of the sample through the capillary. nebulized. This is necessary because large drops do not rema in in the hottest part of the flame for a long time and therefore may not become volatilized and e xcited. before they get into the flame.33 Basic components of an emtssion Jlame photometer (i) Nebultzers or atomizers : Samples. i. The design of a simple nebulizer is shown in Figure 8. . a cloud cham ber is placed between the flame and-the nebulizer where the large drops condense and drain awa y. As the sample emerges from the tip of the capillary it is broken into a mist of fine droplets by the flow of air.34(A). Because of this reason.. To eliminate large drops. must be c onverted into a fine spray.e. nebulizers are essentially of the 'scent spray' type whereby a forced stream of air at nearly the speed of sound) passes over a capillary tube dipping into the test solution.Figure 8.. The large droplet.244 Compressed Sample Drain Figure 8. only 10% reaches the flame) and if one has only a small amount of sample to star t with.34 (B). an improvement on the basic desi gn of the nebIzer has been achieved by placiv an impact bead a few millimeters away from t he nebulizer tip. Moreover. " Compressed Fuel chamber Impact bead Sample Drain Figure 8. there is a large loss of sample (9 0% is lost. Table 8.5): Various gas mixtures producing different flam es differing in their temperatures are used in flame photometry. This design lowers the average droplet size and thus improves the efficiency of the analysis besides reducing the loss of sample.34 (13) Nebulizer with an b'npact bead (it) The flame (Also see Box 8. atleast 2-3 ml of sample are required a .34 (A) Design of a simple nebulizer Since large droplets condense and drain away. To alleviate this problem. consuming. The design of this nebulizer i n shown in Figure 8. when they emerge from the tip.10 lists s ome of the often used combinations. collide with the bead and are broken into smaller droplets. this poses a big problem.. However. (iv) Photocells: These are the usual detectors in a flame photometer.245 Spectrophotometry Table 8.10 Fuels. so dium. Unfortunat ely the flame instability reduces their accuracy. Natural gas Air Nitrous oxide Oxygen Air Air 2400 2800 3140 2000 1500 Mg Ca Na. for routine analysis of such elements as calcium. Therefore a multi-channel polychromato . gases and temperatures used in flame spectrophotometry Fuels Gas (oxidant} Temperature (C} Elements assayed Acetylene Acetylene Acetylene Propane . and potassium a simple filter might suffice. K (it0 Monochromators: In sophisticated instruments prisms or sometimes even diffr action gratings are used. resonance wavelength from a continuous source by using a prism or a diffraction grating or both simultaneously. An important p oint of difference is the need to have a radiation source. Instruments with single and double beam optics are available.r is used in some routine procedures to allow measurement of up to six elements simultaneousl y. In less sophisticated instruments. Instrumentation for Atomic Absorption Spectrophotometry This is practically similar to that of emission flame photometry. It is practically impossible to isolate a particular . The double beam op tical arrangement is more or less similar to that of double beam apparatus in absorpti on spectrophotometry. . Such lamps ha. The lamp will thus emit monochromatic radiation characteristic of the emission spectrum o f the element involved. This problem was solved with the development of hollow-cath ode discharge lamps.e now become commercially available for a long range of elements. a continuous discharge lamp with double monochro mators is used. Such lamps produce monochromatic radiation characteristic of the element analyzed. In these lamps the cathode is a hollow tube which is lined by the element in que stion. copper. iron. copper. plants and even in the macromolecules. soils. Except for minor differences. saliva. mhnganese. tissues. organelles. iron and zinc. plasma. cells. lead and mercury need to be 6xtracted from the biological fluids before they can be assayed. calcium. however.11. It also permits estimation of silver. other body fluids such as urine. lead. selenium. alkallne-earth and rare earth ele ments. Flame photometry. has a very important advantage over AAS i n that it allows simultaneous quantitative multielement analysis to be performed. Flame photometry is very sensitive to the estimation of alkali.Element 246 Bophyscal Cherrtr Applications The primary use of flame photometry and atomic absorption spectrophotometry is i n the assay of elements in biological samples such as blood. A list of wavelengths used and detection limits for various elemerts in emission and absorption flame spectrophotometry is given in Table 8. indium and thallium in a host of biological samples. bismuth.11 Measurement various elements by emission and absorption flame spectrophotomet r: Detection limits and wavelengths used Emission Absor tion Wavelength Detection Wavelength Detection (rim) . potass ium. mercury . Atomic absorption spectrophotometry (AAS) is a more sensitive technique and can detect presence of much less quantities of elements with the exception of alkali and alkali earth metals for which flame photometry is preferred. AAS can detect quantities less than 1 part 10-e of more than twenty elements. aluminium. cadmium. Table 8. Flame photometry is used in routine estimation of sodium. magnesium.limit (rim) limit . gold. the performance of both the techniques is more or less comparable. Although most elemental analysis is possible directly. cerebrospinal fluid and milk. 02 .001 0.5 0. I 0.2 493.7 285.8 372.5 0.3 766.7 324.0 26 0.2 403.(parts 10} (parts 10) Aluminium Barium Cadmium Calcium Cobalt Copper Iron Lead Lithium Magnesium Manganese Nickel Potassium Sodium Zinc 484.7 352.1 0.5 589.005 0.4 326.2 9 0.0 670.1 422. 1 240.4 0.02 .0 0.03 589.001 0.5 0.7 0.9 0.03 213.6 228.3 0.2 283.02 279.5 670.8 1.3 0.05 341.2 0.0.2 248.3 553.0 0.03 422.7 0.02 285.5 0.1 766.0001 309.7 0.5 0. however. Figure 8. Perhaps the greatest advantage of atomic fluorescence is its extreme specificity and complete freedom from many forms of interference. Many physical methods are comparatively uninformative when applied to aqueous solutions. or xenon or mercury discharge lamps are used. It will be observed that a char acteristic feature . A similar relationship exists between atomic absorption spectrophotometry and atomic fluorescence spectrophotometry. Developments in the field of instrumentation will surely make available this technique for use in biology.35 provides a comparison of the magnitude of energies and the frequencies of transitions over the entire spectroscopic range. developments in instrumentation and accumulation of basic data h ave allowed this technique to be applied to determine macromolecular structure and interactions. We may cite the exa mple of zinc and even cadmium where the levels detected are as low as 1 and 2 parts 10-I° respectively. In recent times. are excit ed by an intense source of radiation and their fluorescent emission is assayed at an angl e of 90° in a manner similar to that of spectrofluorimetry. Moreover.Spectrophotometry 24 7 Atomic Fluorescence We have seen the relationship between absorption spectrophotometr" and spectrofluorometry. these atoms. however. however. Lack of sufficiently intense source for many elements has been the limitation of this technique. the flame retains its role as a source of atoms. High intensity hollow-cat hode lamps. and those which can be readily applied often depend on alterations of molecular properties and therefore cannot reflect much of the detailed structural informat ion of interest. NUCLEAR MAGNETIC RESONANCE SPECTROMETRY The use of nuclear magnetic resonance (NMR) in the determination of molecular structure has been a major growth point in biochemistry. Until recently technica l and theoretical difficulties were major inhibitory factors for the application of NM R. the sensitivity of t his technique is much better than that of the absorption method. with time instrumental developments are overcoming this problem. In atomic fluoresce nce. Such spectra can be expected to reflect the more or less unaltered structure and conformation of macromolecules in solution.Radio---EnergyA . that the appearance of the spectra is dep endent on slight variations in electronic configuration. 260 KCal/mole Electronic & Transitions-----.5 x 10-7 Vibrational Rotational ESR NM R . It is natural then.of NMR spectroscopy is the very small value of the energy absorbed in the transi tion of nuclei to the excited state.Vibrational 38 0.-.5 xl0-4 9. Frequency 3 x 1015 5 × 1012 101° I07 4 × 1014 UV & visible ----Infrared -Microwave .56 9. 1. 3S..g. 0. and the electrons possess inherent magnetic fields. Nuclear magnetic resona nce spectrometry addresses itself towards detecting the minute amount of energy abso rbed or emitted as the nuclei jump from one energy level to another. 3/2 . Th e effect of nuclear fields are. .. I. gF.. 2H. 9/2 depending on the particular nucleus.Nuclear magnetic moment (Magnetic component of I7' . N). Figure 8. Artificially created intense magnetic fields can. Nuclei possessing anodd mass number {either the number of neutrons or the number of protons should be od d.al Chemistry Most nuclei. too small to be observed in the ambient magnetic fi eld of the earth. the charge on them is distributed non-symmetrically (e.. Such a nucleus is then supposed t o possess an angular momentum represented by a spin quantum number. There is an associated nuclear magnetic moment. which is assigned half integral values. Nuclei possessing odd numbers of both neutrons and protons hav e an integral spin number of I = 1. This spin ning charge gives rise to a magnetic field.. but never both odd) are assigned half-integral spin quantum numbers. experiments because they do not possess an angular momentum (I = 0) an d do not exhibit magnetic properties.r. O.) ar e not measured in n.m. etc. 1/2. P(½) and B(3/2).36 (A) illustrates a nuclei as a spinning sphere. Nuclei possessing even numbers of both protons and neutrons (2C. for example H. however. along the axis of the spin. North magnet pole H ---. make the nuclei assum e specific orientations with corresponding potential energy levels.. the protons.. Magnetic Properties of the Nucleus In order to explain the magnetic properties of certain nuclei it is necessary to assume that the nuclear charge is spinning around an axis. Thus... however.248 Biophys. 36 (A) Spinning charge in nuclei generates a magnetlc fleld with a rnagn etc moment p. On ly that RF whose sense of rotatlon is e. When a second. precess ab out the fleld direction in such a way that each pole of the nuclear axis sweeps out a c'cular path in the XY-plane . like the top of a gyroscope.field of rad frequency (RF} is applted at right angles to the uniform mag netic fleld. . the nuclei may undergo a transttion to a higher energy level by absorbing the RF energy. When placed in a powerful unorm magnetlc fleld the spin axes of these nuclei align themselves in an manner with respect to the fleld and. weaker.mctly equal to the rate of precesslon of nuclear dpole re quency of RF equal to precesslon frequency) will be absorbed.Precessional axis South magnet pole / lgure 8. These two levels (Figure 8. gF and sip. I-2 -I Any given nucleus can have 2I + 1 possible levels or orientationsl Thus for IH.36(B)) will have the energy of -Ho a nd +Ho for low and high energy respectively. In a magnetic field of several hundred millitesla . This is given by the series : l. only two orientations are available. is then equ al to 2Ho.zE = hv = HoI I = -V2 Alignment with the field Figure 8. E. The difference in energy. The value of I determines the number of q uantized energy levels available. such nuclei must absorb the appropriate quantum of energy.36 (B) Energy states for nuclei in a magnetic fleld In order to change from low energy state to high energy state.1. where Ho is the intensity of the applied m agnetic field and is the nuclear magnetic moment.Spectrophotometry 249 Magnetic nuclei assume discrete orientations with corresponding energy levels un der the influence of external magnetic fields. Ho . with I = V2. energy).Ho/I Alignment against the field External field. 15N. and (tO antiparallel to the field (higher energy). The general relationship is as follows : E -.I. These levels are described as (0 aligned with the applied field (lower. I= +V2 T No field <. m. . The relationship obviousl y describes the ratio between the frequency and the field. The above equation can. also be written as AE = hv -. thus.2 ]Ho YHo hi here 2 serves to convert linear frequency to angular units of frequency.2Ho (for cases where I = If we combine the two equations. This phenomenon is known as nuclear resonance or nuclear magnetic resonance. Another mathematical expression for ¥ can be derived from the above relat ionships : Gyromagnetic ratio =y This relationship is a fundamental equation for n. we get 2rv . ¥.r. The ratio is sometimes termed *re sonance condition'. which is obviously equal to 2r/hI is known as the gyromagnetic ratW and is a characteristic propert y of the nucleus.(several thousadd gauss} such nuclei absorb radiation in the radiowave region of the electromagnet ic spectrum. the peak corresponding to CH3 is much higher than the peak co rresponding to ---OH.r.37 again.r.. Figure 8. is dictated by () the isot ope being studied. let us consider the number of signals generated by some compounds as examples. Acetone andcyclobutane are considered first. spectrum of methanol (CHaOH) Obviously all nuclei do not absorb at the same applied field but the absorption depends upon the magnetic field which a particular nucleus. For a clearer understanding. Such a plot is shown in Figure 8. viz. at a g. . The structur al formulae are given below.ven radio frequency'. and (tO the intensity of the magnetic field. dlfferent nuclei will require slightly different magnetic field strength to give rise to the same effective magnetic strength which causes absorption. the effective field strength is different for different nuclei as one nucleus will have slightly different environment from any other nucleus. in Figure 8. therefore. The number of signals obtained on such a pl ot are indicative of the different sets of equivalent nuclei or protons.m. a normal practice to measure the absorbance of a sample at a monochromatic radiowave frequency while varying the magnetic field intensity. Thus. applied.OH Acetone Cyclobutane 250 Btophyslcal Chemistry The frequency of the radiowave absorbed during n. It is. CH3. For an example one ca n refer to Figure 8. n. Clearly. 37 there are two signals which are indicative of two sets of protons existing in different enviro nment.37. spectra are thus a plot of energy absorbed against the magnetic field str ength. The area under the peak in such plots is directly proportional to the number of nuclei or protons which are responsible for the resonance. For example. and --OH. feels.r.m.37 The n.m. i. a given set of protons is chemically equivalent only if all the protons exist in identic al environment even when stereochemical formula of the molecule is written. signals in the following manner: . on the other hand has three sets of equivalent protons existing in three different environments (CH3. and --CHO). We have already seen how CH3OH gives two n.r. signals owing to two different s ets of proton equivalents. CH3CHBr2. signals.e. l-dibromomethne.r. in an n. --CH.2-dichloropropane is expected to giv e three n. This principle can be gr asped better with the help of an example. Propanal.Hsc/C ---0 CH----CH It is easy to see that all six protons of acetone and all eight protons of cyclo butane exist in the same environment. Another compound Which gives two n. only one signal will b e observed for each of these compounds.r. experiment.m. Chemically equivalent protons should also be stereochemically equivalent.r. The compound 1. sets of protons existing in two different environments .m.m. Thus.m. signals is 1. this compound gives three n.m.. Therefore.r. CH3CH2 CHO. The area under the peak of cyclobutane will be more tha n that under acetone peak owing to a larger number of protons in the former. m. An applied magnetic field. Each o the above named sets of protons have electronic environment characteristic of the type: These proton sets theref ore absorb at different applied field strengths. Chemical : Position of Signals We have seen that the number of signals in an n. Consequently these two protons will give rise to two different signals rather than one. circulation of electrons around which has g iven rise to a magnetic field. This magnetic field is prop ortional to the applied magnetic field.c. The electrons involved in covalent bonding are paired and usually do not have a magnetic field.r. however creates additional modes of c irculation of these electrons thereby generating a small localized magnetic field.r. acetyleni. To understand this le t us take a look at the stereochemlcal formula of the compound.oH° 3 2 i CHz--CH(CI)--CH2CI In an n. experiment. The phenomenon is known as d/amagnet/c shie (Figure . Cl 1 1. Such nuclei. spectrum gives information about the number of equivalent sets of protons/nuclei that the compound possesse s. vinylic. aliphatic. this compound gives four signals. or whether the proton ex ists near an electron withdrawing group or an electron releasing group. might not resonate. the position of the signals can reveal whether the environment of proton is aromatic.m. It may be said that the electronic environmen t of the protons dictates where the proton will absorb in the spectrum.2-Dichloropropane It is evident from the above formula that the two protons attached to C do not e xist in exactly identical environments.251 Heft : Ho . For example. The position of these signals is indicative of the nature of the environment surroun ding the protons/nuclei. 8. Thus. a higher magnetic field will be needed to make the nuclei reson ate. and the nuclei are said to be shie/ded.. shielding of nucleus proton shifts the position of the signal. they are experiencing a smaller magnetic field due to the oppeelflon created by the field of electrons. It is thus a function of the structure of the compou nd. where ¢ is the shielding constant whose Caue depends upon the density of electrons around a proton or nucleus.kL . H0. For the above situation. Such nuclei are not resonating be cause rather "than experiencing the fuji magnetic field applied. Le.38). absorp tion upfle. The proton is therefore deshielded and requires lesser magnetic field to resonate.. This iS because in the former.38 Diamagnetic shielding. For proton spectra in nonaqueous media. Local magneticJlelds opposing Ho generated by uuced electronic drculatlon Magnitude of shielding decreases with increasing electron withdrawing power of t he nearby substituents (strongly electronegative substituents such as oxygen).-: :.F:. P rotons in CH3 are more shielded and therefore absorb at a higher magnetic field.. It is usual. The signal due to CH is therefore shifted upfield. """ ?. TMS contains 12 protons and all these protons are chemically equivalent. Sometimes t he induced field augments the applied field. the reference m aterial is tetramethyl sflane (CH3)4Si.r. The nucleus is then said to be deshlelded and a smaller applied field is required to make such nuclei resonate.-. just as we didin case of absorption spectrophotometry. deshielding shifts the abs orption downfleld.37.m.252 Induced magnetic field Ho -.i Electron circulation SnPulcenlulslg Figure 8. the proton is adjacent to the strongly electron withdrawing oxyge n atom.m. to compare t he positions of n. Therefore.r. spectrum of CH30H given in Figure 8. absorptions arising out of shielding and deshielding with a standard r eference. Thus.r. Let u s again consider the n. One can see that the absorption due to --OH is occurring at an applied field lesser than that required for --CH3 . The above discussion can be understood better with the help of an example. abbreviated as TMS. TMS gives a single sharp signal in .. absorptions as compared to the standard refere nce are known as chem/ca/sh/fls.m. The shifts in positions of n.'.. tl e positions of the absorption lines for the sampl e and reference respectlvcly. n. signal for tetramethylsfla ne is taken as a reference and chemical shifts for different sets of protons is determined rela tive to it. experiment. In comparison to this reference TMS signal. it may be said that shielding of equivalent protons of TMS is greater than most of the organic compounds. Most chemical shifts have values between 0 to 10.m.r. and i is the opera ting frequency of the spectrometer. This difference in the absorption posttlon of tte pro ton with respect to TMS signal is called chemical shift. signals for different types of protons will appear at different field strengths.m. Therefore. The magnitude of the chemical shift Is expr essed as 8-value which is calculated as follows: 8 = HSAMPUS --HTMS X 10e where HSAMPLE and HTMs are .r.n. Silicon has a very low electronegativity and therefore equivalent protons of tet ramethyl sflane are deshielded very little. is usually expressed in parts per million {ppm}. expressed in frequency units {hertz}. . m. Protons which experience greater deshielding have larger values. TMS Highly shielded signals ] I0 9 8 7 6r 5 4 3 2 I 0 of protons Primal> Hydroxyl Esters Acids Acetylinic Chlorides Ethers Alcohols Fluorides Vinylic Aromatic Aldehydic Carboxylic Phenolic Enollc R-CH3 Chemical shift in ppm . A list of values for chemical shifts for protons in diff erent environments is provided in Table 8.39}." It is usual to plot n. signals with magnetic field strength increasing to th e right.imple relationship: = 10.Spectrophotometrtj 253 Another scale used for measurement of chemical shift is known as the x (tau) sca le. TMS is assigned a value of 0 ppm and its signal appears at the extreme right (Figure 8.12. This is so with most of the organic compounds. It is related to scale by the following s.r. 0 .9 1.5 4.0 8.2 2.0 9.0 3.44.02.5 2.02.8 6.03.0 3.1 9.5 3.R-OH H-C-COOR H-C-COOH CmC-H H-C-CI H-C-OR H-C-OH H-C-F C--C-H Ar-H RCHO RCOOH At-OH C=C-OH 0.5 2.65.0 4.O4.34.04.05.0 10.09.0 8.0 9. 0 6.0 6.17.12.8 7.0 5.6 6.0 6.O 10.5 7.5 6.0 to -5.0 to -0.5 to +6.0 5.7 6.0 15.4 4.5 6.0 .0 1.0 4.5 4.0 7.2 1.0 .12.0 0.5 4.0 -2.0 .0 .8. 0 .0 -7.-2. ese ee ways of coup e 8. Consider eyl brode. i. a b CH CH Br It Is erefore et at e n.ways in relation to the external field as outlined below.).. Let us now consider the reason for a quartet signal for the-CH2 group.m. We have dy seen how elg d deselg ect posion of n. spectm of this compound.0 4. e n.0 3. e sp of (HO c couple e ney meylene oup ee disct ways reon to e e fi eld..e.0 6.m. i.r.r.0.0 7. s of s md Ist of is. however. (re 8.0.0 5. The three protons of the methyl group couple with the adjacent methylene group in four di fferent. a cluster of pes. specWa. speca bec ome fuer compcated what Is o as spin-sp coup.0 1.m. we ow at s molece cons o ds of protons denot as a d b low. ''"- (A} (Relnforcmg) Signal from - {B) (Not effectlng) 'a' protons - (C} . 2. We so eect t e o sis be smets. .r.e.m.(Opposing) Spin influence of protons 'b' This is the reason why a triplet signal is given by --CHs. We m dersd s phenomenon fly e foHo phs.r. e sis each have one peak. y have e ss spt? e molece o f eyl brode. For a up of ptons we obsee a slgn consisting of a cluster of three pes (plet) whereas e s for b oup of protons consis of a cluster of fo ps (quiet). show s two sisals compos of mple. CHCHBr: from what we have considered so f.External field 254 Biophysical Chemistry S Uttlng . e n. it is clear that the slittLng of the signals into multlplets has been because 'the different environments of two sets of protons with respect to the nearby pr otons. a triplet while the other is a doublet.0 .i Spectrophotometry 255 /(A) . however. This which involves interaction between like and different spins of nearby nuclel is Analysis of the n. We again take up the the first example. we ex pect its to be deshielded more than that of the methyl group.m.6 as compa red | that for the methyl group which occurs at 8. CI2CHCH2CI.41 shows two signals indeed. We are. at a stage where me e. (ReinforcLng : Strong) .s.0 e. but both the signals are multiplets. (B} l (Reinforcing : Weak) External Slgnal from (C} 6' 4% (Opposing : Weakl field 'b' protons (D) (Opposing : Strong) Spin influence of protons 'a' From the above.aa y to a given compound gives a partlcular n. figure it can be seen that the signal for methylene group occurs at 6.r. Consequently we ex pect the due to the methylene group at a lower applied field as compared to the methyl gr oup. Which of the slgnal belongs to aand &O ?. Figure 8.r. Since the methylene group is nearer to the bromine atom.O . spectrum of a compound. From its formula we can deduce that the compound two signals. It does. b a As a second example let us consider Lelchloroethane.. only the empirical formula for wh ich is beyond the scope of thls book. tw o sets present and we might expect two signals. One signal is a quart et We have already seen the particular spin-spin coupling by which we that the quartet belongs to the methylene group while the triplet belongs the methyl group.m. It is that this mutual magnetic influence between the two sets of protons is not trans mltted but through the electrons of the chemlcal bond between the two groups. spectrum.As indlctted.35 ¢. rnlght expect prot on set more d6shielded as compared to o. If this is so. therefore.0 9. See text for descr/pKon (taken from cata/og ue). the triplet which is present at a lower should belong to group b and the doublet should be due to a.1) 1.0 U . We.0 0 ppm (6) :.0 3. Let us also confirm this by .41 N'MR specan of bdoroethane.4.0 4.0 8. to b? The proton set b has two chlorine atoms (electron withdrawing) near it proton set ahas only one such atom near to it.0 Hgu 8.0 7.0 :3.0 2. m.r. high speed digital computer.m.m.m. In compar atively old instruments extremely heavy electromagnets (103 104 kg) produced fields ranging from 1-10 tesla. Instruments was much less as compared to other optical techniq ues and such other techniques as gas chromatography. it is a difficult Job to interpret n.r.co Chrrtrv consideng the spin-spin coupling. It would not b e wrong to say that the magnets are the heart of an n. instruments.m.m. Due to all the complications which split and shift the peaks. in organic chemistry. On the other hand signal from proton b will be split by proton set into a triplet in the foll owing manner.r. The newer gendration spectrometers.5 teslas. called the Fourier transform n.r. however. (Non effecting) [ External (C] (Opposing) | field Thus from spin-spin coupling also we can see that the triplet belongs to b while the doublet belongs to o. The magnetic field can be vari ed by .r. Sensitivit y of the old generation n. and mass spectrometry. These instruments have revolutionized the practice of n.Bophus. spectrometer. are butt around a sm all. The mode m day n.r. Splitting of signals from proton set aby the p roton b should as follows (A) ' (Reinforcing) T External (B) (Opposing) I field Thus coupling of proton set a with a single proton b will give rise to a doublet . use superconductin g solenoids which generate fields above 3. spectra. spectrometer. (A) (Reinforcing) (B) . Instrtunentation Figure 8.m.r.42 shows the basic components of an n. Usually services of an expert are required. Since the nuclei absorb in the radiowave region. Sample is usually placed n a high precision diameter tube (this minim izes the variations in the magnetic field) and is rotated at high speed by an air turbine .42 Main components of a nuclear magnetic resonance spectrometer Appl/eations "(0 Structural diagnosis: The n. . structural informa tion which . Thus. Sample Radio transmitter / . The absorption signal is then detected by a radio receiver.about 10-2 tesla by using an auxiliary sweep generator.The signal is amplified and recorded.r.m. the source of radiation is a radio frequency transmitter.. The sample must be dissolved to a relatively high concentration in a solvent which is proto n-poor or CDCI3). spectroscopy is mainly applied to study the structure of small organic molecules and small globular proteins./ Radio receiver Amplifier Recorder /weep coils Sweep Magnet generator Figure 8. n. Extensive use of n. an agonist to the receptor.r.r. has also been u sed to measure intracellular pH. has been used to study complex formation su ch as the binding of a ligand to an enzyme.m. Measurement of the phosphocreatine concentration in muscle can be cite d as a representative example. . ha s been made to study the conformation of the lipid headgroups of the biological membran es and their interaction with integral proteins of the membrane. and calcium bi nding proteins have been obtained using n. As a direct method n. studies. cytochrome b5. prothrombin.r.r. Another example can be given of the measurement of hexakisphosphatephytic acid which accumulates in the pith tissue of plants.m. In the recent past n. has been used to determine the concentration of metabolites. affect the intrinsic properties of n. (iii) Studies on complex formation : Using n.r. spectroscopy. aromatic side chain rotatio n.m. hen egg-white lysozyme.r. n.the internal motions of macromolecules or chemical exchange. spectroscopy.m.m. a drug to DNA. or an antigen to an antibody. various cytochromes. howe ver..m. (iv) Biological structures and compartments : Compartmentation is an essential p art of the organization that characterizes living systems. resonances and make it possible to pro be the dynamic aspect of molecular structure by this technique. plasminogen and chromogranin A. and overall tumbling of protein have been st udied using n.r.m..m. The proteins which have been studied for their dynami c characteristics include histones. internal motions of proteins such as the opening of secondary structure.r.r. has an undoubted advantage over freeze -clamping analysis.Spectrophotometry 25 7 relates to the biological functions of the antibiotics such as valinomycin and g ramicidin have been obtained from n. (v) Quant/tat/ve stud/es : Over the years n.m.m. Thus. Structural information about small prote ins such as some neurotoxins.m. and metabolite concentrations. For protein studies. interconversions and fluxes. ha s been increasingly used to study this biological structure.g. segmental motion of the main chain. membrane transport phenomena.r. it is possible to detect very small conformati0nal changes. (ii) Study of dynamic-characteristics of protein structure: Processes which modu late intemuclear distances.r. Thus. the technique is combined with X-ray tudy for better information. e. . m.g.m.al muscle. The techniques has also shed welcome light' on the mechanism of action of proteases and on the mechanism of their inhibition by som e proteins.r. m. can be used to measure the associated thermodynamic quantities.r. and partition coefficients for the distribution of a molecule between different compartments.r. Examples can be cited .: In one of the most intriguing new areas in biochemical n.r. The technique has been used to study alanine and lactate transport in the human erythrocyte by exploiting the d ifference in the magnetic susceptibility between the inside and outside of the cells. haemog lobin) has been determined using n. pKa of histidine residues in proteins (e. n. 31p has been applied to the study of such intact biological specimens as heart. response of the two states is different.r.r. and skelet..m.m. heats and entropies of binding.r.of studies made on folding and unfolding of proteins and tRNAs.r. pKa values. This has been possible largely because o f the development of instrumentation capable of resolving 31p resonances of small mole cules .(vi) Thermodynamic studies : In the general case of an equilibrium between two s taes. n.m. and the interac tion between actinomycin D and deoxy-pGpC.m. Very recently Na÷ transport in human erythrocytes has also been studied using n. (viii) Intact organ studies with n.m. kidney. has been used to measure quantit ies such as binding constants. if the n. (vii) Membrane transport: Membrane transport either in in vivo systems or using synthetic membranes has been studied using n. s. pe eas of e sple e comped to e pe ea of a said wch conts a o quflW of unped elecWons. e complemen tecques. howler.m. stentation ure 8. Monocomac owave radiaon t be read y obted y us a yson oscator. AE. e mec field is ved e resonce occas. elds of 0 llltesla. EON SP NCE SPERORY A chec species odd number of elecons ibits chactesc meflc propees much e e nucle us.m.) e appropte qut of ener is obned from radiaon e crowave re. P resonce stues of ceH metates is now prodg sit to ceHul compenflon. from e above it t en at n. elecon a ec field is able to absorb ener of e proper frequent.s. () : Bioloc objects such as its. .eae phosphate. nuts d ce have n ed proton n.s. sc. In a mer si to e ato c nucleus. e e. specWa.r. If an intense magnet ic field is applied.r.on of e elecoec has en seen respect to n. adenosine diosphe. are enerated by elecomaets.r.. g.s. d orgc phosphate -.43 usates e basic components of e. a mec field of e oer of gauss (i for e.m. be 2 Ho. is phenomenon is o as ecn reso. specometer. e pK's of er sin molecules of phys ioloc impoce e ound 7.r.s. us their 3p chemlc shifts e sensive to pH d c be used to mease WaceH pH. pe is fly propoon to e numr of ped elecons e sple vesflgated d us to e concenWaon ofe ple. HJ or st (er ener.r.r.668.s. + HJ e rid& e ener derence. d e. speca do ebit he sptg wch is caused by tem¢ons een e sg elecWons d adjacent spg malefic nuclei. e e.r.r.e moles most pot ceHul physiolo.r.r. In qumflve ysis. the electron assumes oenmons ower ener. e spng acon of unped elecon generates a maec moment.m.r. = wch catapult it from e lower to a hier ener level.599.s.r. ple yon urce t p cs Oaor Met .. required for accurate ork. zeuatoaphy. A sweep enerors a capacl of I0-I00 tesla e so prodded.) s. e ea of e. Le n.629258 Bphsat Chemtr such as adenosine triphosphate. show no phenomenon wch is pel to e chec s seen n. d e tecque wch is employed to study s e of behaor is ced eon sp reso (e. It is coon practice to subject e sple to deg male fic tensies keepg e crowave frequen const.590.m. I for studies with steroids. haemoglobin. and iron (cytochromes. fatty acids and phospholipids. Thus. molybdenum (xanthine oxidase).44).4 4(A)) were developed between 1960-65. Electron spin resonance spectrometry is one of the main methods to study transit ion metal containing metalloproteins. superoxide d ismutase). In addition to the nitroxlde moiety.259 Saml)les for e.r. While some proteins or enzymes contain intrinsic functional groups with unpaired electrons.e. ferredoxin.. Appliemtions Electron spin resonance spectrometry has proven most helpful in studying mechani sms of reactions which proceed through free radical intermediates. Figure 8. or C--N bond.s. i. C--S. biological macromolecules which lack unpaired electrons can not be stu died by e. Biological samples which cort laxi[e mouv. Spin labelling refers to the use of stable free radicals as reporter groups or labels.44 Tgpa:al spia labels .s. experiment. mxst be soids.r. These macromolecules can however be "spin l abelled" (spin labelling involves the attachment of a stable and unreactive free radical to the macromolecule) and studied. lateral diffusion of glycerophosphatides in plasma membranes has been studied by labelling glycerophosphaUdes with a stable nitroxide free radical. Another class of nitroxides developed in 1967 (Figure 8. these labels contain a fun ctional group useful in covalent attachment of the nitroxide moiety to a larger molecule throu gh a C--O. The term "spin label" was first coined by McConnell and co-workers in 1965. Spin labels (stable free radic als) are usually molecules containing the nitroxlde molety that contains an unpaired electron loc alized on the i: nitrogen and oxygen atoms (Figure 8. because they do not resonate. Adjacent methyl radicals (Figure 8.t o i water are therefore frozen in liquid nitrogen before e. copper (cytochrome oxidase.44(B )). there are many which do not. e tc.r.s. known as *doxyl" are usefu. Much information can be gamered about these macromo lecules by incorporating in them an extrinsic functional group with unpaired electrons.) Normally. (Figure 8. and phosphonylating (Figure 8..45 (A)). ff they bind covalenfly are spin/abe/s. other labels. substrate-inhibi tor analog spin labels carry those functional groups which have been deliberately designed to L at specific high affinity site(s) on the macromolecule and/or covalenfly bind with hyperreactlve group in the macromolecule structure. affinity label study.45 (B)). These labels might be classified as alkylaing.The biochemical methodology behind designing a spin-label experiment is analogou s to for any other reporter group study (e.g. The bound labels. For pr otein a large number of covalenfly binding labels with specificity for distinct amino acids become available lately. binding noncovalenfly are known as spin probes. acylatin g. . In addition.r. (tii) Denaturation and protein folding: If a spin label is present in an envir onment which is sensitive to the degree of folding or unfolding of the overall protein structure . In fact. salt. even subtle ones. These strategically placed labels also yield information about protein-protein interactions. much information is reported by it about the denaturation and renaturation process. O ver the years these studies have yielded much important information about the struct ural features of these conformational states. s pin labels have been shown to be excellent reporters of conformational changes.6). (i) Rates of catalysis: The rate of some step in an enzyme catalyzed reactio n can be under circumstances where a change in motional state of the spin label is exhibi ted before and after the reaction.s. spect . or temperature-induced structural changes. spin labels bound at the active slte provide much information about the enzyme-ligand interactions. {B) Phosphonylating spin labels.r . A brief list of the kind of information that spin labels can provide in an e. (ii) Active site geometry: Use of spin labels has provided much information a bout the depth or width of the substrate binding region of various enzymes. experiment is given below (also see Box 8. ThuS.260 Biophysicat Chemistry [A) (B} llure 8. (iv) Enzyme-ligand interactions: Depending on the environment of placement. e. these labels provide information about the pH.s. One of the ve ry important applications has involved the use of spin labels to compare the active site conformations of families of related enzymes. This is possible when (i) the conformational of the spin label on the protein is sensitive to steps in catalytic mechanism.45 {A) Sulfonylattng spin labels. a nd/or when the spin label is (one oi) the substrate. (vt) Spin labels have provided insight into the kinetics and mechanism of str uctural changes of a variety of condensed phases of DNA. Such studies are currently in progress and are expe cted to .rum of a spin labeled protein easily detects the narrow line spectra exhibited by proteol ytically damaged species comprising a few percent of the total protein. Spin labeled enzyme crystals provide informatio n about their symmetry and orientation. (V) Symmetry and orientation studies: Studies of proteins by spin labeling m ay be applied to crystalline proteins as well. (vii) Studies of hydrocarbon and phospholipid motion in lipid bflayers have be en performed with the aid of spin labels. a brief explanat ion and a brief idea about the applications of this technique is given below. Similarly. As compared to controls. (Ix) Other muscle diseases: Work with spin labels is helping in providing ins ight about the molecular nature of many muscle diseases. Studies with spin labels. With the of which is about 2. some may be introduced into biological macromolecules by the so called enrich ment Mossbauer isotopes most often used for biological applications are 5rFe. Discovered by Rudolph Mossbauer in 1957. have studied erythrocyte membranes from human patients affected with myotonic dystrophy using fatty acid spin labels as probes . suggest abnormal calcium pumping in diseased membranes and altered interactions with primary membrane proteins such as the calcium ATPase.(vaO 261 provide much Information about the domains of phosphollplds involved in these motions.2% in abundance in the natural isotopic mixture of iron . and 2I. the label was found to reside in a more fluid less polar l ocus in patients. We have seen that atoms can transit between excited and ground states by absorbi ng or emitting electromagnetic radiations in a manner dictated by their electronic str ucture. It has thus been concluded that myotonic dystrophy is a diffuse membra ne disorder. isotopes occur in high proportion in biom01ecules. the phenomenon a technique which has since had numerous applications in biophysics. nuclei also are capable of transitlons between certain nuclear energy levels as a consequence of . However. when taken together with other measurements. Myotonic dystrophy: Roses eL a/. It is present a complete analysis of this phenomenon in this sectlon. MOSSBAUER SOPHOTOMETRY The Mossbauer effect is based on the resonant absorption of nuclear gamma radiat ions by certain stable nuclear isotopes. is the energy of the emitted photon. . c is the velocity of light. T he recoil of nucleus is an energy requiring process and is defined by the following relations hip 2 RE = E/2Mca where M is the nuclear mass. whenever a T-ray is emitted form a nucleus. E is less than the transition energy AE. Since both E and RE are derived from the energy of transition. E. and RE is the recoil energy.absorption or emission of electromagnetic radiations such as -rays. In order tha t momentum be conserved. the nucleus recoils. the emitted y-ray will have roughly the same ene rgy as the.----. This became possible because the recoil energy in these cases was not su fficient to make the nucleus move as a free particle.46.4 KeV EI y-ray El ---------G 14.4 KeV --. energy of transition and therefore will be absorbed by another atom of the same type: Thus. and the former is used as a radiation source in studies of latter . 57Fe is the Mossbauer isotope of keen biological interest because of the natural occurrence of iron in a number of proteins such as cytochromes. Absorption of this energy raises another Fe nucleus . This phenomenon is known as the Mossbauer effect and the technique based on this is . The momentum of recoil in these cases was t ransferred to the entire molecule in which the nucleus was bound. G and E are the. haemoglobin.ground and first excited nuclear states respectively. myoglobin. the emi tted photon would have possessed exactly the same quantum of energy wch could be absorbed by another nucleus of the same type. Figure 8. We will therefore discuss Mossbauer spectroscopy with respect to this isotope. due to the energy loss.lG STFe Fe Figure 8. In such cases. fer redoxin etc. As shown In.4 KeV y-ray. Probabflttles of t he steps aregen tnpercentaBes. 5C0 decays in two steps. S Co is the parent nucleus of Fe. when y-absorption is measured as a function of energy of the incident radiation. a very sharp resonance absorption line is seen at the transition energy of Mossbauer transiti on.46 Schematic representation of EC decay ofTCo toTFe. E to G tra nsition results in the emission of 14. Mossbauer discovered exce ptions to the above phenomenon when he found that certain low energy y-emisslons took place wi thout any recoil. and it can not be further absorbed by another nucleus. However.262 Biophysical Cherrdstry If the nucleus had not lost some of the transition energy in the recoil. called Mossbauer spectroscopy.. and the large mass of the mo lecule protected any energy loss. the emitted photon ha s less energy. STCo 14. It is interesting to note how the range of energies is produc ed. and over a small range of energies on either s ide of the transition energy. the energy E of the y-ray varying by a small amountAE according to the relation AE . #. Co is used as the radiation source.4 KeV energy is moved by an electromagnetic drive system at const ant velocity. it gets converted.from G E state.4 KeV. the EC decay does not lead directly into the ground state o f SFe. A typical measurement requires velocities o f the order . The absorption of gamma radiation produced by cobalt-57 source by Fe in the sample being studie d is then measured at various temperatures. to the stable daughter nucleus Fe.f energy that ground state SFe can absorb. O. toward and away from the sample (absorber}. Since this is the exact quantum o. which emits 14. This movement results in a Dopple r effect. in the first step. This state has a mean life of just 140 nanoseconds from which it descends to the ground state by recoil-free emission of gamma ray photon of 14. Through EC decay (see chapter 13). The source. However. rather it proceeds through the first excited state of TFe which is located 14.4 KeV abo ve the ground state. is.positive when the source is m oved toward the sample and negative when moved away.(0tc] E where cs the velocity of lght. The velocity. rather.5 Doppler velocity (cm/sec) Figure 8.48. An electromagnetic drive system (EDS) moves the energy source (S) towards and away. Doppler s hift velocity is used for this purpose. Al l these effects depend on temperature and just like in NMR (where different kinds of splitting p rovided information about the environment) these effects. The band may become further split by what is known as hyperflne or quadrupole splitting. Moreover. Lead collimators (C) dej'be beam geometry. In the discussion above we have said that 'a very sharp resonance line is seen a t the transition energy of Mossbauer transition'. This is known as isomer shift. As an example.47 Principle of Mossbauer spectroscopy.48. From Figure 8.0 1. For ex ample. since they are produced by the local environment.5 0 0.5 1. It should be noted that the energy of incident radiation u sed in a Mossbauer spectrum is not expressed in terms of wavelength or frequency. This local environment is responsible fo r broadening the absorption line into a band of variable line width.from the absorber (A) with a constant velocity i. it become s clear that this is not so and in effect quite a few bands are seen.48 Mossbauer spectrum of human methemoglobln cyanide . This is so because the characteristic features of a Mossbauer spectrum are affected by the local environment in which the nucleus giving rise to Mossbauer effect exists. Mossbauer spectrum for methemog lobin is shown in Figure 8. -1.47) and this as a function of the Dop pler velocity/) is called the Mossbauer spectrum. provide much information about the local environment itself. The y-ray intensity transmitted by the sample is recorded in a pro portional counter placed behind the sample (Figure 8.Spectrophotometry 263 of some mm/s. however.0 -0.5 -I. the band may be shifted slightly in energy relative to the standard energy source. Ttansmltt ed radiation is measured by the proponal counter (Z. Figure 8. but also in the lattice around it. these effects tell us quite a good deal about the interactions taking place not only w ithin the nucleus itself. such studies have proved very sensit ive in distinguishing different Classes of cytochromes. .Most of the applications of Mossbauer spectroscopy to biology have been with res pect to the iron containing proteins. Therefore. For instance. one electron is transferred to the oxygen molecule. Nu merous publications discuss the mechanism of reversible binding of an O2 molecule to th e iron in haemoglobin. the electro n configuration of better described as an Fe3+O configuration. rather than as Fe2+O2. Yet. However. Mossbauer studies o n oxygenated haemoglobin have provided some evidence that duing the binding of oxy gen to iron. the most studied iron containing protein has been haemoglobin and some results of such studies are cited here. the mechanism is not understood in detail. The details of the process are beyond the scope of th is book and it will suffice to say that the technique is used to solve phase problems in X-ray structure determination of proteins. Can you find out how much oxidized and how much reduced form of t he coenzyme is currently present in the tube? Let us tell you that only NADH absorb . it is situated in a pocket a nd oxygen reaches the iron via a channel which leads from the molecular surface to the heme pocket .D. to which ox ygen binds is not situated at the surface of haemoglobin. Iron.both NAD+ and NADH. of 0. Apart from these studies. Now the solution contains. the change i n the affinity of haemoglobin for oxygen consequent to pH changes must be due to changes in the molecule occurring at greater distances.4 at 260 nm. Mossbauer studies have provided quite a lot of information about the dynamic pro perties of biomolecules.2 264 Biophysical Chemistry Mossbauer studies have also provided some data on the Bohr effect (chapter 1) wh ich speaks about the regulation of oxygen affinity of human haemoglobin via pH chang es in its surroundings. Let us again take the example of haemoglobin. Such changes are probably alloste ric in nature. Mossbauer spectroscopy has provided a good deal of inf ormation about the electron structure of bacterial ferredoxin. What Mossbauer studies tell us is that the Bohr effect may not be due to any change in the immediate surroundings of the iron. This solution gives an O. Although the inference drawn by such studies is negative. A tube containing NADH in solution was exposed to. X-ray diffraction studies tell us that this channel is too narrow for the passag e of oxygen. How does the oxygen reach iron? Recent Mossbauer experiments give direct evidence th at the protein molecule indulges in a sort of breathing motion which opens the channel momentar ily wide enough for the oxygen molecule to pass through. rather. Some Solved Problems I. Mossbauer spectroscopy is now being used along with X-ray diffraction for struct ure determination of proteins.5 at 340 nm and 1. oxidizing atmosphere by mistake. it never theless will be helpful in the ultimate explanation.1. from the iron. 22 x 103 Thus concentration of NADH is 8 × 10-5 M The O.5 = (6.000 at 260 nm and 6220 at 340 nm.D. The extinction coefficient of NAD÷ at 260 nm is 18.22 x 103) (1)(C) 5x10-I C =0.8x10 6.s at 340 nm. Calculating NADH concentration from O.Let us find out just how much abs orbance is due to NADH.D. A = ambC am for NADH at 260 nm is 15 × 103 A = [!5x I0){I)[8 × 10) = 120 × 10-2 . at 340 nm is uncomplicated sinc e NAD÷ does not absorb here. at 260 nm is due to both NAD÷ and NADH.0 00. Also assume that the cuvette used has a standard path length. The extinction coefficients of NADH are 1 5. A = abC 0. s. but both NAD÷ and NADH absorb at 260 nm.. out of the total O. The mol ar absorption coefficients for Z are 7000 and 6500 at the two wavelengths.5 x 10Cz) Solving for Co in terms of Cz (You may do the opposite and yet solve the problem ) 0. = am be 0.225 = 15xlO3 Spectrophotometry 265 Thus.36 = 15 x I03C+7 x l0sCz . 1.000 and 3000 at 450 nm and 600 nm respectively. The solution contains two substances G and Z. Let us write equations for O.m = (am 450(G} x Co) + (am 450(Z) + C} 0.D.0. 0. O. Both the substances absorb at 450 nm and 600 nm. The molar absor ption coefficients of G are 15. It is now easier to find out the concentration of NAD.Ds at both the wavelengths first.225 = (3 × 10Co)+(6.11x10-4 18x103 Therefore.225. of only 0 .m = {am 600(G) × Co) + (am 600 (Z) × Cz) 0.2 is due to NAD÷. concentration of NAD÷ is 1.36 = (15 x l0s Co)+(7 x 103Cz) Aoo. of 1.4 at 260 nm. The absorbance at 450 nm is 0.1 × 10-s M.36 and at 600 nm is 0. Can you calculate t he concentration of the two substances? Assume that you are using :a standard pathlength cuvette.225 = A4o.D.2 = (18 x 10s)(1){C) C = 2xI0-1 = 0.2 is due to NADH. 36.225 -15x103 Multiplying the right hand term with 15 x 103 6.5x 108Cz . at 600 nm 0.5 x 10sCz) 0.225 -.D.7 x I03C Z 15x103 Substituting the above value of CQ in the equation for O.36-7 × 103Cz = 15 × I03Ca 0.5x103Cz 15xi03 1.0.08×103 .5x103Cz + 15xlOa 1.08xI0 -21x108Cz 0.(3x103) 0"36-7xi03 Cz +6.21x lOaCz + 97.225 = (3 x 10CG)+(6. Assume that you are using 1 cm path-length.D.3x103 .0 ml of solution containing excess A TP. The molar absorption coefficient of NADPH is 6220.266 3. Glucose + ATP Glucose-6-phosphate + ATP Glucose-6-phosphate dehydrogenase reaction : . NADP.91 after a considerable amount of time had elapse d.450 or 600 r im. and glucose-6-phosphate dchydrogenase. Assumption : The two enzymes added to the tube will carry out the ollowing react ions : Hexokinase reactions : . 0.0 ml solution of glucose was added 2.15/15 x 103 = 1 x 10-SM Therefore the concentration of G is 1 x 10-5 M.5 x 108Cz 2.08 x 103-21 x 10sCz + 97. Given that K values for hexokinase and glucose-6-phosphate dehydrogenase lie far to th e right. The absorbance at 340 nm due to NADPH was found to be 0. We may now substitute the concentration of Z in the equation of O.38 x l03= 1. MgCI2.15 = 15 X 103 CG C = 0.36 = 15 x 10CG + 0. Can you calculate the concentration of glucose in (i) the final. and (ii) the origin al solution.21 0. To a 4. hexokinase.3 x103 =76:5x 103Cz 2.36 = 15 x 103 Ca + (7 x 103) (3 x 10-) 0.5x10s Therefore concentration of Z is 3 x 10-5 M. .3xI0-SM Cz 76.36 = (15 x 103C) + (7 x 103Cz) 0. one mole of NADPH will be gener ated. C (original) = C (final) x dilution factor The dflutlon factor of the solution will be 6/4.22 × 103)(I)(C) 0.M NADPH Since we are taking NADPH as an index of glucose concentration. Therefore it can be safely assumed that for every mole of glucose consumed. For finding out the concentratio n of glucose in the original solution. The absorption at 340 nm is solely due toNADPH and glucose and other components of t he reaction mixture do not absorb at this wavelength. measuring NADPH will mean measuring glucose. A = ambC 0.Glucose-6-phosphate + NADP* 6-phosphogluconic acid--lactone + NADPH + H÷ The Kq values are far to the right and ATP and NADP* are in exdess.91 6. .91 = (6. the above is als o the concentration of glucose in the final solution. we will have to take into cognizance the dilution factor.463 × 10.22 × 103 CNDpH = 1. or 3/2. since one mole of NADPH is be ing generated for one mole of glucose consumed. However. I ml of the preparation. can the s ame scheme of things operate? Also.M = 6. * Instead of glucose. However.43 × 10-3 units of the enzyme are present in 0. and (ii) the spe cific activity of the enzyme. only 0. Thus the effective concentration of protei n was reduced to 5 mg/ml.Ix moles/ml/min. Activity = aA/min = 0. The assay mixture of a total volume of 1. 0.1 ml of enzyme was present in I ml of the assay mixture.1 ml of enzyme preparation.463 x I0-) x 3/2 C (origlnal) = 2. Calculate (i} the units of the enzyme present in 1 ml enzyme preparation.0 ml contained excess lactate.43 IX moles/litre Activity = 6.43x 10.43 × I 0-2 S.43 × 10.2 × 10-4 M.43 × I0. if one has to measure glucose-6-phosphate.04/min A = (am) (aC} {b} AA (am)(b) = 6.units. C (original) = (1. Therefore 10 times the amo unt will be present in 1 ml of the preparation. 6. The enzyme preparation contained 50 mg protein/ ml. Semicarbazide is also added to the solutio n with a view of capturing pyruvate and carrying the reaction to completion.43 IX moles/lit/min = 6. The enzyme preparation contains 50 mg protein/ml. = 5 . The absorbance at 340 nm increased at a rate of 0. buffer. Therefore the specific activity of the enzyme here was 6. What reaction mixture can you prepare for measuring the concentration of glucose-l-phosphate? 4. study the metabolic fate of glucosc-lphosphate.A. generation of NADH in a 1 cm cuvette spectrophotometrically.Spectrophotometry 267 Therefore. Thus 1 ml will contain 6.04/min. Lactate dehydrogenase was assayed by measuring the. Brown.. 1975. Modem Optical Methods of Analysls.. Willard. 1980.B. Instrumental Methods of Analysls. E. H.013 units/mg protein. and Settle..= 0. 1986. Merr/tt. Butterworths. C.N.. 1975 . 4. O]sen. McGraw-Hill.H.A.. An Introduction to Spectroscopy for Biochemists.A. Wadsworth Publishing Company. S. USA. London. New York. Rao. Ultraviolet and Visible Spectroscopy. Academic Press. L. J.R.D. F. London.. Dean. 3. Suggestions For Further Reading Ultraviolet and Visible Spectrophotometry I.L. 2. . 2. ESR I. and Herrmann.. .. S. "C. Rao. Fundamentals of Analytical lame Spectroscopy.. Chemical Applications oflnfrured Spectroscopy. London.H.V. Pinta.L. S. 4. 3. London.B. Willard. N. and Settle. New York.L. 3.E. Infrared Spectrophotometry in Methods in Enzymology. Fluorescence Assay in B{ology and Medicine. USA. 1. . 1969. Udenfr/en. C. 1979.A. New York. 1967. Wood. 2.A. Academic Press. Alkemade. Daly. Introduction to Infrared a nd Raman Spectroscopy. Wadsworth Publishing Company.0 Fluorescence and Phosphorescence of Proteins and Nucleic Ac ids.. D. Academic Press. 2. H. Ne w York. F.. Instrumental Methods of Analysis. 1957. 1986.Biophysical Chemistry Fluorescence 1.cademic Press. 3. Ne w York. S.. 1975. Dean.2. and Wiberley.. M. L. Infrared Spectrophotometry I. 1975. Konev. Vol. IV . Hflger. Flame Emission and Atomic Absorption Spectrophotometry 1. . Colthup. J. 1963.. Hilger.H. L. Plenum Press. Bristol. R. Academic Press..N. Atomic Absorption Spectrometry.. Merritt. M. Science. Ann. Construction and Evaluatlon of aSelf-Luminescent Biosensor. Spin Labeling in Enzymology : Spin-Labeled Enzymes and Proteins in Methods in Enzymology. J. Vol. and Patel..]. and GriiTith. 2. Holt. Osgood. 1992. Science. London. Campbell and R. London.C. 3 King. Rechnitz. Nuclear Magnetic Resonance Spectroscopy in Physical Prtnc/ples a nd Techniques of Protein Chemistry.. L. Moore. London. Meritt. A.54 at 500 nm. F. Exerclse I.J.. M. D. Instrumental Methods o f Analysis. Part B. O. A. and Settle.D. 1978. Vol. and Haschemeyer. S. Vol. Vol. Metcalfe. New York.K.. Academic Press. 19 83. News.R. 1977.A.H.S. Luminometry I. Acad.L. New York.. 227 : 778-781. 1990. Wadsworth Publishing Company. USA...Y.H.A. Academic Press. 1988.M.C.62 at 475 nm and 0.P. Substances A and B absorb at both 475 nm and 500 nm.. Academic Press. Vol.J. Edinburgh : Churchill Livingstone. 66 : 24-36. Chem. 1.. R. L. 1970.E. H.N. N. R. 646 : 53-59.. John Wiley.G.A. Compound B has an a of 50 . and Holmes. ad Williams. of 0. A. J.J. Marshall.2. T he a of " compound A at 475 nm is 12000 and at 500 nm is 4000. R. BiologicaI Nuclear Magnetic Resonanc Spectroscopy. NMR and the Biochemist in Essa ys in Biochemistry (P.H. Ratcliffe. G. Academic Press. P.. XLIX. Berliner. Haschemeyer. (1985) in Recent Advances in Cl inical Biochemistry. 1978. 3. J... 198 : 158-165. 1973. eL al... Dean. XLIX.D. Prote/ns.E.. Opella. Sci.H.. Willard.. 3. G. Vol. L. 4. p. Geiselhart. The Spin-Labeling Technique in Methods in Enzyrno logy. Jost. These substances ar e given to you in a single solution which shows an O. Eng. i986. Campbell. Eds. cuvette used to take the reading has a path length of I cm. Calculate the concentrations of the two substances i f the.00 at 475 nm and 11600 at 500 nm. . The enzymes glutamate-oxaloacetate transminase (GOT) is assayed by a cou pled system. On the basis of the above solve the following problem.D. A reacUon mixture containing excess asparate.D.5 ml of a solution containing the enzyme glutathione reductase and an unk nown concentration of oxidized glutathione.glutamate + oloacetate (b} oxaloacetate + NADH malate + NAD÷ (the first reaction is catalyzed by GOT and the second by malate dehydrogenase).3 x 10-MNADH (b) 9 x 10-2MNADPH (c) 2. was added an equal volume of a solution w hich was 2x 10 M with respect to NADPH. (a) 7. First calculate the O.9 ml.25 in a 1 cm c uvette.21 x 10-2 units/ml Calculate the O.11 (c) 0.[B] = 0. Large excess of NADH is added which gives a good O. the rate o f decrease of O.3 } mole of NADH. Since both the reactions are going on simultaneously. having the following reactions. The O.37 x I0-M To a 1.5 ml of the solution? a of NADPH at 3 40 nm is 6220.1 ml. (Hint. The second reac tion depletes NADH depending upon the concentration of oxaloacetate generated in the first reaction. The cuvette has a path length of 2 cm . 3. is driven by the rate of appearance of . 0. 3. at 340 nm.27 Make a study of glucose metabolism and on the basis of your study describe a pro bable .42 x I0-aM. The absorbanc e decreases at the rate of 0. (a) aspartate + a-ketoglutarate----.2 x 10-MNADH (a) 0. and an excess of malic dehydrogenase in a total volume of 0. can you calcul ate the concentration of oxidized glutathione In 1. Calculate the concentration of GOT in the serum as unltsml.D.M in the original solution.196 x 10. The reaction is started by adding excess of a-ketoglutarate in 0. at 340 nm stabilized at 0.oxaloacetate and is therefore th e index of GOT activity. of the following soluUons at 340 nm in a 2 cm cuvette.91 (b) 0.D.1 ml serum (which contains GOT). Given that the glutathione reductase reaction is as under Oxidized glutathion + NADPH + H÷ reduced glutathione + NADP* and that the reaction proceeds completely to the right hand side. 0.269 [A] =0.D. of NADPH due to the given concentration ) Concentration of oxidized glutathione = 0.04 unit/rain. DNA from B s hows a corresponding increase in O. beginning at 65°C and plateaus out at 76°C. A and B.method for spectrophotometric measurement of giucoose-l-phosphate based on light absorption at 340 nm. 6. Inadve rtantly. D NA from A shows an increase in optical density beginning at 70°C and a plateau at 80°C. You have prepared two concentrated solutions adenine and guanine.D. the values also return to the origin al values. You have two samples of DNA derived from different organisms. There is Just one phenylalantne residue in a given protein which is abso rbing maximally at 259 nm with an a¢.ack to 100% water. What is your conclusion? . What is y our conclusion? 8. Can spectrophotometry resolve the confusion? 7. If the protein is now dissolved in 70% water and 3 0% glycerol the wavelength of maximal absorpUon changes to 265 nm and extlncUon goes up to 2 17. you have forgotten to label the flasks and now you are not sure which flask contains what . of 206 (both of which are very near the normal values) if the p rotein has been dissolved in 100% water. If the protein is brought b. Discuss factors which give flse to fluorescence.15. fluorescence is obned. 11.s e ee cted. However.1 M NaCI. If the concenaflon ofe protein is cased to 5 mg/ml 0. so e acid titration er decreases tenai. if eylene glycol Is added now at is hi ptein concentration. At a concenton of 55 mg/ml ff eylene #ycol is added to e soluon to a level of 3/0 go e mmum absoflon velen d e ecflon increase as eomped to what ey were in 0. e intensi of fluorescence is inordinately low. at foaon c be obned from e n absoflon s? 17. what consideraOons me e conclusion invMid? 14. at mgement may be present some specophotometers If e eors due to voltage fluctuaons e to avoided togethe Discuss. 18. at is met by e te chec sh? WHte eples. fluoence decreases. 16. More th one elaon is possible). at do you conclude? er. Yet. Biolhysica Cherrstnj A protein is o to possess a se oph d a se phenyle residue. A ven protein conns oy one toph which is shong a fluorescence mum as at for flee toph. acid flaflon of e protein tes you at e pK of sion is due to a cbol. en S Is bound to a ven prote. 12. Descb¢ briefly e eo of n specome. A given protein is o to con 12 tophs. If not. at do you ow about is toph no -. at conclusions may you draw about e prote ? I0 at is absoflon mum ? Does e sctur chmcter d the posion of absoflon mum depends sole upon e scture of e compound ? Discuss. at foaflon is prodded by e positions of sign. Your coen? (Note. ff e prote concenaflon is incased fore addg S. each of e foOt. g o e X d e encflon increase Howler. y e fluorescence spec bd sp ec ? 13. no fuer specM chges e seen.g: . I M NaCI.270 9. is it vd to conclude at ree of e tophan residues e on e surface. If cesium long quench 25% of e fluorescence. e shielding d deshieldg effects i nvolved n specome.s specm? How my sign. (c) buol d (d} eyl acetate? . ) eol.(a) prope. 1). The observable result of this preferential refraction is a rotation of the plane of polarization since the angle a and no longer remain identical. since a light wave consists of an electric and a magnetic component vibrating at right angles to each other. considered to be a sum of two circularly polarized components (Figure 9. A plane polarized b eam is.1). This wavelength dependence. of the op tical rotation is known as optical rotatory dispersion. This difference between the indices of refraction of the medium for the r ight-hand and the left-hand ray results in retardation of one component more than the other. therefore. where n = the refractive index. This unpolarized light can be resolved by Nicol prism into two beams in which al l the rays vibrate in only one particular plane. (i) An anisotropic medium can refract the right (R) and left (L) components of the plane polarized light to a different extent so that I'll nR.cted light from any light source behaves as if it consisted of a large number of electromagnetic waves vibrating in all possible orientations around the direc tion of propagation. This rotat ion of the plane of polarization might be due to either of the two phenomena considered below. the term 'plane' in not so right. This phenomenon is known as optical rotation. but the ray can be said to be plan ar if the meaning is restricted to convey the position of the electrical component only). (//) If the plane polarized ray has the wavelength falling within the range o . it would be seen that the plane of polarization has been rotated (Figure 9.9 OTHER OPTICAL TECHNIQUES FOR MOLECULAR CHARACTERIZATION CIRCULAR DICHROISM AND OPTICAL ROTATORY DISPERSION Natural unrefle. If the medium preferentially r efracts the left circularly polarized component the resultant would be a plane p olarized ray i'otated somewhat to the right from its original position (Figure 9. The extent and even the sign of op tical rotation differs at different wavelengths. These beams are known as plane polarized ( of course.1). Each one of the plane polarized beams emerging out of the Nicol prism can be further resolved into two beams of circularly polarized rays by a device known as Pockels cell. the right and the left circularly polarized components. and vice versa. If we pass these circularly polarized rays of light through an anisotropic mediu m. f the absorption maxima of the anisotropic medium. the medium will absorb it. however. . The optically activ e medium. shows a preference for absorbing either the left or the right c ircularly polarized component. its transmitted portion will have less intensity than the incident ray . In the previous case of optical rotation due to refraction both the refracted co mponents had equal intensity. however. The resultant light is said to be e///pt/ca//y po/ar/z ed (Figure 9. since one of the components is being preferentially absorbed. Figure 9. In this case. but the two transmitte d components will also differ in intensity.272 b I I I I (B) Figure 9. in this case. not only will the plane of polarization be rotated.2]. a denotes optical rotation. The plane of polarized l@ has been rotated due to retardation of one of the ciradar component s. EL (s the left and (B) Result of the passage of plane-polarlzed light through an optically acti ve meal:lure. It i s coJculated from the following relatlonshtp: .2 Elliptlcalpolarlzatn oftlght upon leaving an optically active medium in the absorption range of an optleally active troJitWn` Note that E is shorter than E. The dierential absorbance of the circularly polarized light resultlng from a differe nce in absorpttvtty of the two rays is called circular dichroism (CD).1 (A) Representation of a plane-polar&ed ray as a sum of two clrcularly polarlzed rays. Thus. 8 describes the CD ellpiaj. respectively. it is only for the sake of understanding that they are being discussed independently). If this s so. one can apply Beer's law to circular dichrosm. (0 Extinction coeffcients are always positive. The circular dlchrolsm spectrmn may be thought of as the difference between two absorption spectra. we get AL-A = (aL--aR) Cb. Thus AL = aLCb and A = aCb where the two subscripts L and R indicate the results obtained employing the lef t and right circular components. optical rotatory dispersion an d circular dichrolsm are mathematically commutable so that one can be known from the value of other. Taking the difference. As circular dichroism depends only in those spectral regions where absorption occurs will not be observed in the regions where no absorption occurs.. a = -aR = -AICb extinction coeffcients. that is aa isomer = . In the latter sp ectral aL and a will be zero. one obtained using the left while the other using the right circularly polarized component. . The usual representation spectrum is to demonstrate Aa as a function of wavelength.. Thus. There are two major differences between CD spectra and absorption spectr a.Other Optical Techniques for Molecular CharacterizatWn 273 We will now discuss the theory behind circular dichrolsm and optical rotatory di spersion individually (as we will see in subsequent pages. However.a isomer (/0 . optical i somers of any given compound at a given wavelength will give rise to CD spectra of opposit e signs. Aa can assume eithe r a positive or a negative sign depending on the relative sizes of aL and aR. In actual practice a plane polarized beam is passed through quarter-wave plate which is rotated from +45° to -45° producing first d and then I c ireularly measures the periodicvariation in the transmittance : when the incident radiation experiences periodic changes in the handedness. (aL + a/2. aa for most of the molecules of biol0gical in terest are less than I% of (aL + From the second point above..CD spectra and the absorption spectra differ in the relative size of a a nd the average extinction coefficient. ff we use the relationship between A and. we can understand that the value of Aa is very smal l. we get a = (-log TL + log TICb = l/Cb log TR = log Is/I° T. Cb I.T. How do we measure it to get the CD spectrum? Ideal situation would be to fill a cons tant pathcell with a solution of particular concentration C and then measure absorbance d ue to left and right components (AL and A) individually and then arrive at the differe nce one from another. (A = -log T)./Io = log Iz Cb IL . Those o f ha on the other hand are centimeter squared per mfllimole (cm21mmole). if t he sample shows circular dichroism. and IR are the intensities transmitted. is given by b a = 180 -(I'll .. This is so since refractive indices are never zero even in regions where the sample is t ransparent (does not absorb). If the sample does not exhibit circular dichroism. If the specifi . The light path b is exp ressed in decimeters and the concentration C is expressed in grams/100 cm3. the common form in which optical rotation is reported is either the spe cific rotation [a]. Optical rotation.2). The circular dichroism of the sample is determined from the magnitude of the oscillation in the intensity of the transmitted beam s triking the detector. On the other hand. circular dichroism is also expressed in terms of molar ellipticity [0].(3300 deg mmole/dmole) Aa Optical Rotatory Dispersion In contrast to circular dichroism.nR) However. The units of [0] are degree-centimeter squared per decimole (deg-cm21dmole).274 Biophysical Chemistry where I. Since the projections of the electric vectors in circular dichroism differ not o nly in their angular velocity but also in length (Figure 9. the oscillation frequency of the transmittance will be same as the oscfllaUon for the handedness of the incident beam. Thus even those solutions which do not absorb in the ultraviolet re gion wig glve optical rotation in these regions. Due to this. [0] -. where [(] = 100 alCb. a. The following equat ion relates and Aa. where(m) = [a] M /100. TR and TL will be the same and log (TR/TL) will be zero. The units for the specific rotation are deg-cm/decagram. or the molar rotation (m'). an optically active sample gives rise to opti cal rotation even at wavelengths which do not fall within the region(s) in which the sample a bsorbs. the polarization of light leav ing the medium is elliptical. CD and ORD Spectra Figure 9. Note that the units for [M'] and [0] are identical. circular dichroism and optical rotatory dispersio n spectra for a hypothetical optically active substance which has a single absorption band .. the ORD spectnzm shows a more complicated pattern. but maximum optical rotation is obtained at wavelengths slightly away from . .3 depicts absorption. Th e similarity between the absorption and CD spectra wig become obvious from the figure. is denoted by a dashed line. If measurements of op tical rotation are limited to these regions which are far removed from the absorption bands. Even those regions which are far removed from Zm. On the other hand. Th is property of opUcal rotatory dispersion is very useful for compounds which absorb maximally a t vacuum UV where determination of CD is tedious. =. The wavelength at which the maximum absorption takes place. for such compounds optical rotatory dis persion can be measured at wavelengths far removed from the vacuum UV. x exhibit some optical rotation. no optical rotation is observed at X. the molar rotaUon is described by the Drude Equation.c rotation is multiplied by molecular weight M and divided by 100. . it gives rise to molar rota tion whose units are deg-cm/decimole.=. Experim ental determination of [M'] at different wavelengths would give rise to optical rotato ry dispersion spectrum. Absorption maxlmo are denoted by the dashed vertical lines. Rearranging the above equation we get .lm'l Other Optical Techniques for Molecular Character/zat/on 9.275 O.2 [m'] -.:[m']versus[m'] will be a s traight llne.3(B) depicts the absorption. where K and o are constants. Note that while the absorption spectra in both cases are identical. It is interesting to note that the ORD curve resembles the first derivative of t he CD curve. (A) and (B) denote spectra of c ompounds which are rrdrror brcu3es of each other. Further dLscusskm n the text. " Figure 9. It s assumed that the compound has only one absorption band. circular dichroism and optical rot atory dispersion spectra of the mirror image of the hypothetical molecule considered in F igure 9.. clrcular dichrolsm and optical rotatory dispersion spe ctra for a hypothetical compotmd which s optk:ally active. If the equation does not make a fit. In fact the above equation fits optical rotation of transparent aqueous so lutions of numerous biological compounds in the visible region of the spectrum. the circular d ichroism and optical rotatory dispersion spectra have changed signs If a racemic mixture of t his hypothetical compound is subjected to the above experiment. [m'] + K It would seem from the above equation that a plot oD. ORD and CD are therefore coupled phenomena which in principle are mathematically .3(/I). the absorption spectnm wo uld still be identical but the circular dichroism and optical rotatory dispersion spectra would cancel out.3 Idealized absorption. the inference usually is that the visible optical rotation of the biological sam ple is due to contribution from more than one optically active absorption band. commutable. Inte rpretation . It is possible to calculate CD spectrum of a given compound from its ORD spectrum by applying the mathematical relationship known as Kronig-Kramer transform. ORD and more preferably in modem days.276 BiophysPzd ChemLstry of ORD spectrum is made difficult by the fact that it is complicated and extends to regions far removed from . for example proteins are made up o f L-amino acids and these residues would contribute to optical activity.7.. At this pH the e-amino groups are protonated. an asymmetric di stribution of charges or dipoles about a chromophore. To understand how measurement of optical activity can provide us an idea about t he conformation of a macromolecule. the constituents of the macromolecule. In all these cases spectropolarimetry comes handy. X-ray analysis might be virt ually impossible if sufficient quantity of the material is not available or if the molecular weig ht is too high or if crystallization is a problem. Although. This is especially so for those molecules which have multiple o ptically active absorption bands. X-ray diffraction crystallography is the method of choice for structure determination of biopolymers. This synthetic polymer is readily soluble in water at pH 5. Consequently the polymer can exist in a variety of confor mations. Applications (1) CD/ORD of Proteins . if one determines the optical activity of a given macromolecule. an a nalysis will provide good information about the general structure of that macromolecule. The . Besides. Obviously. Optical ativity of a macromolecule is usually a resultant of three factors. spectropolarimetry continu es to make important specific and complementary contributions. these r esidues might be arranged in an ordered fashion such as the DNA double helix or the a-helices of proteins. For this reason circular dichroism. we will consider an example of synthetic polype ptide poly-Llysine. is preferred. Thirdly. which is easier to interpr et. as prevalent in a rigid tertiary structu re. In t he first place. will also give rise to optical activity. CD spectra are used to deduce the chain conformation o f macromolecules.. Secondly. and this arrangement is also a basis for optical activity. by modem standards. this method consumes very little time as compared to X-ray analysis which is an extre mely lengthy procedure. 4(A)) of this random cot (only CD is discussed . observe. Obser ve the CD (Figure 9. . however. There is a we ak positive circular dichroism at 217 nm and a strong negative circular dichroism at 197 nm. the CD and ORD undergo drastic change. Negative circ ular dichroism is now seen at 208 and 222 nm and a strong positive circular dichroism is seen a t 191 nm. ORD is zero). the polymer adopts an ordered structure known as the a-helix.4(B)) and ORD (Figure 9. Again. Observe th e drastic change in the CD and ORD as a consequence to this confo. that wherever extinction is maximum for CD. If one removes the positive charge of z-amino groups by raising the pH of the so lution to 1 I. not much change in the absorption spectrum is observed which ex hibits only one absorption maximum for all conformations at i90-200 urn.rmational change. 1. A positi ve circular dichroism is now seen at 195 nm and a negative band is seen at 217 nm. It may be mentioned that while all these drastic changes take place in the optical activity as a con sequence of changes in conformation.polymer may then be thought as existing in a random cot state in solution. The synthetic polypeptide can be converted to the -pleat sheet structure by heating at 52°C for 15 minutes and then cooling. 1969. N. From X-ray diffraction studies we know that whale myoglobin exists largely as an a-helix (about 80% of the amino acids are p resent eight segments of a-helix). Figure 9. Descriptn in text.) Let us see whether the same pattern of optical activity is found for other prote ins of conformation. ioo% 3.. sperm whale myoglob in phosphate buffer pH 6..277 -20 Other Optical Techniques for Molecular Characterization [m'] x 10 CURVE A I I.250 9. I00% a Helix Rdom coil 190200 210 220 230240250 CURVE' I. 100% Random coil 19o 200 'i0 220 230 240.40pti6al rotatory dispersn (A) and circular dichroism (B) of poly-L-lysine in the a-helicczi random coil. 8:4 108. Greenfield. 100% a Helix 2. Biochemistry. G. 200 220 WAVELENGTH {nm) L20 . and -conformatins.86.D .5 depicts CD spectra of two proteins. (Adapted from. and Fasman. . W. 15 : 4264. mochomtry. Maitre.86. oi. (B) Concanavaltn A n phosphate buffer (curve I) and concanavaln A n phosphate bu ffer + 0. Descrtn m tex (Adapted from.018 M sodan d. pH 6.ajl sulphate (coe 2). 1976) . and (B) eoneanava lm A.L et.5 C'radar dhrolsm spectra for (A) sperm whale myoglobln.240 .-0 220 240 WAVELENGTH (rm) 9. (A) Sperm whale myoglobin in phosphate buffer. H owever. The CD of pure enzyme is shown in Figure 9.278 Bophysk Cherrrlstrg Observe that its CD spectrum shows the same qualitative features as observed for a-helical poly-L-lysine. One of the substrate for it was p-hydroxybenzoate. Description tn text (Adopted from Teng. . it was not definite whether NADH. eL al. The results of the ex periment are shown in Figure 9. X-ray diffraction studies confirm that there is no a-helix in concanavalLn A and about 50% of the amino acids adopt the -plea t sheet structure.spectra was obtained for the enzyme in the presence of NADH and NADPH.02 ENZYME + NADPH | Figure 9. The CD spectrum for concanavalin A has the same qualitative featu res as that of poly-L-lysine -pleat sheet structure (Figure 9. We can infer that the detergent sodium dodecyl sulphate has induced formaUon of a-helix in co ncanavalin ConformaUonal changes in the enzyme do take place when a substrate interacts wit h it. on the enzyme p-hydroxyb enzoate hydroxylase.al. It can therefo re be concluded that the enzyme specifically binds NADPH which must be the coenzyme used in" the reaction.. N. From the results it is clear that upon NADH addition th e CD spectrmn of the enzyme solution does not change significantly. A case in point is the study carried out by Teng et. or NADPH was the other coenzyme needed. BioL Chem. It was known that the enzyme has a tightly bound FAD moiety. T herefore CD and ORD measurements provide a sensitive method of studying enzyme substrate complexes.. whereas upon NADPH additio n the CD spectrum of the enzyme solution undergoes significant alteration.. The spectrum shows the same pattern.6.6 Effect of NADPH on CD spectrum of the enzyme p-hydroxybenzoate tujdro lase.as expected of an a-helix.6. A CD. 246 : 5448-5453. J.5 also depicts CD spectrum of cocanavalin A upon addition of sodium dodecyl sulphate. Figure 9.5(B)). i ENZYME + NADH 0. 1971). We have already seen that CD and ORD are responsive to conformational changes. CD and ORD remain important methods to study the structure of proteins.5. Moreover. This is the reasonwhy CD and ORD are extensively used to study conf ormational changes in proteins and polypeptides. solvent effects might also play a large role. With al l these ambiguities. Many protei ns which have been shown to exist largely as a-helices do not show the qualitative featur es of a-helix shown in Figures 9. changes in optical activity may not be t otally assignable to conformational changes. .4. 9. be pointed Out that rel ationship between ORD. however. CO.Figures 9.4 and 9.5 and 9. It might.6 show that CD and ORD are responsive to conformational c hanges of proteins. and chain conformation is not as simple as we hope it tobe. An extremely important application of CD is its use as a 'finger-printlng' techn ique for qualitative analysis of trace amounts of carbohydrate materials such as mucopoly saccharides. and position of substitution in carbohydrates. The CD spectra of these derivat ives is shown in Figure 9.4 CH2OR CH20R OR " OR (B) a. Chain conformations of a-and -llnked glycan derivatives have been studied throug h CO. OR CHOR OR IAI 6-1. The s ctra for these two mucopolysaccharides differ from each other .8 shows the CD spectra of dermatan sulphate (a mucopolysaccharide containing 1 .. CD can indicate the nature. whereas the -linkedcellulose derivative did not. The studies suggested that x-linked amylose tricarbanilate adopted a hellcal con formation. Wtly. extent. Vol.s Cell Biology. I. 9.4 G.-7 220 240 260 Other Optical Techniques for Molecular Characterization 279 (2 CDORD of Carbhydrate CD and ORD are also frequently used in studies on carbohydrates. Frequent use of CD has been made to study not only the conformati on of other polysaccharides but also their association in certain polysaccharide gels like a lginate and pectin. Perhaps the best example of such studies is afforded by studies on oligosaccharides from blood-group substan ces and human milk.R.3sugar) and heparitin sulphate (a mucopolysaccharlde containing 1.4-1inked sugar).7. 1973).1. in New Techniques n Bphys. ccharldes.8CD spectra of two mv. 9.R. Vol. (Adapted from Morris.R. in New in Bhyslcs ¢md Cell BWlogy. Wiley...I01 ----Dermatan Sulphate 8 /! Heparifln Sulphate Fre. and Sanderson.copolyso. 1973) . G. E. I. (3) CD/ORD of Nucleic Acids Over the years. the most important contributo rs are the bases : the purines and the pyrimidines. However. pectin in the presence of citric acid an d sucrose).al Chemtry significantly and this difference lends itself to an important clinical applicat ion. if after some preparation the urine is sub jected to CD. Here it is extreme ly useful because it can measure a very small amount of optically active polysaccharide present in a large excess of optically inactive substances (e. Since the bases contribute to the CD signal owing to the chiral ity induced by their attachment to ribose. an identification is readily made. Therefore when we measure CD of nucleic acids. However. Their optical activity increases further when they assu me helical structures. In themselves. they become optically active when attached to a ribose su gar by means of an N-glycosidic bond.. a negative signal whereas the pyrimidine bases have a positive signal. The ribose phospIiate backbone of nucleic acids does not absorb significantly ab ove 180 nm.4x I0" 2x 10 -4x I0 DenatUred 28o BphsP. An accumulation of these two polysaccharides occurs in the urine of patients with the Hurler syn drome. CD and ORD have also been extensively used in the study of nucle ic acids. the purine bases have. The syndrome has two variants: the Sanfllippo variant where the primary polysaechari de accumulated is heparitin sulphate and the Hurler variant where a mixture of dermatan and hep aritin sulphate is excreted. Through chemical means it is very difficult to identify the two muc opolysaccharides as separate from each other. both these kinds of base s are symmetrical chromophores. the base composition of a given nucleic acid should . CD has also been used as a quantitative metho d for estimating optically active polysaccharides such as mucopolysaccharides.g. At 270 nm. polyadenyl(c add [poly (A)] and denature d poly (A). this contribution is almost insignifican t when compared to the contribution made by the orientation of the bases. It does also. How sensitive this probe is will become clear ff one compare s it with simple . CD (and of course ORD) is therefore an extremely sensitive probe of conformation al changes in polynucleotides. It is this stacking that contributes most to the CD spectrum of the nucleic acids.280 300 (rim) Figure 9.. We know that the bases are stacked over each other. -2 x I0 .contribute to the CD spectrum. CD spectra therefore give us more information about the conformation of n ucleic acids rather than their composition. However.9 CD spectrum of adenylc acid.Ac acid poly (A) 240 260 . Explanation n the text. etc. A few examples of the use of CD/ORD in studying nucleic acids are provided below . of B-DNA is D/fferent as Compared to A-DNA B-DNA is the native form of DNA. ionic strength. Since it is such a fine probe. There are I0 bases per turn of the spectrum of B-DNA is shown in Figure 9. helical twist per base pair is 36° and the pitch is 34°. pH. we find that the latter absorbs 26% less than the form er. Again note the large difference in the native and the denatured sp ectra. The diff erence between the CD spectra of the two is much larger than the 26% decrease in absorption. Note the negative band centered aroun d nm and a positive band around 275 nm. what this means is that the DNA in our cells is in this Outside the cell.10. CD/ORD is used to study (1) si ngle-doublestrand transition of nucleic acids.. If we study the absorption of light of 258 nm by adenylic acid and by polyadcnylic acid. -I0 -15 . (2) denaturation of nucleic acids due to var ious agents such as temperature. The hypochromicity is due to the formation of an ordered structure by the polymer. The zero occurs at about 258 nm. It is exactly due to such extreme differences that CD is such a sensitive probe of cha nges in nucleic acid conformation. and (3) binding of proteins. we can obtain B-DNA when the humidity is 92% and the is an alkali metal such as Na÷. other sma ll organic compounds as well as cations. Al so shown in the same figure is the change that occurs in the CD spectrum of polyadenylic aci d upon denaturation.9} of adenylic acid and polyadcnylic acid. This is a right handed helix with a diameter o{20A .I0 5 Other Optical Techniques for Molecular Characterization 281 absorption spectrometric studies. N ow take a look at the CD spectrum {Figure 9. Formaldehyde is a compound that disrupts hydrogen bo nding. T his high value means that poly(C) is very asymmetric. It may therefore be as sumed duplex RNA assume the A-form.160 deg M-Icm-./ in has 11 bases per turn of helix. Curiously. and a pi tch of This DNA shows an intense positive band at 190 nm. Base-Stacking ORD spectrum of polycytidylic acid shows that the [m]292 is 35. the samecharacteristlc is given by duplex RNA. A-DNA is 26 . In other words this means that it i s helical. The that suplort this helicity can also be determined easily. I0). has a helical twist of 33° per base pair. Addition of formaldeh yde does change [m]29 significantly. a good negative band at 210 n m. I0 CD of B-DNA.. Explanation in the text this with the CD of A-DNA (not shown in the Figure 9. and positive band at 260 nm. were a function of hydrogen bonding between bases. formaldehyde addition should .210 230 250 270 290 310 Wavelength (nm) Figure 9. the CD band for DNA shows about half the maximum ellipticity and half the area of the spectrum for free DNA. The instrument possesses the following basic parts: (i) A light source . we know that the DNA CD spectrum is mainly due to base stacking. The point is co nfirmed by X-ray diffraction studies which give the same result. ORD spectra therefore provide a very good support for base stacking. When bound to histones. Therefore. On the other hand. and the concentrations of the interactin g species. the chromati n spectrum may be telling us that the bases . X-ray diffraction is a far better tool to obtain detailed structural information . As a result of CD studies we know that DNA interacts with different histones differen tly and with different conformational changes in each case.282 Biophysical Chemistry have reduced [rn]292 considerably. Ethylene glycol disrupts interaction bet ween hydrophobic groups.are less stacked in chromatin. the [m]29 is found to be 7223 deg M-cm-. A reduction by such a substance means that the helix was mainly supporte d by hydrophobic associations between the bases. when ORD spectrum is determined when poly(C) is suspended in 90% ethylene glycol. However. From the example about base stacking given ab ove. X-ray diffraction studies are not such a good idea when we want to study the DNA-histone interaction depen dence upon such factors as the pH. ionic strength. CD scores over the former in that it is rapid and can give reliable information abo ut DNA-histone interaction as well as about the parameters that affect this interaction. DNA has a Different Conformation in Chromatin Does DNA have a different conformation in chromatin as compared to the conformat ion in the free state? It would appear so from the X-ray diffraction studies and the CD studies. Instrumentation for Measuring Optical Rotatory Dispersion Optical rotatory dispersion is measured by a spectropolarimeter. This is a huge reducti on and it means loss of asymmetry or of helicity. Since this did not happen we believe that hyd rogen bonding does not contribute significantly to the helicity of the polynucleotide. for example sodium lamp emits only two-589. simple filters are required (this is because these two lamps emit l ight of a limited number of wavelengths.e. In these instruments. In more sophisticated instruments. or a polaroid filter. However. the plane polarized light emitted by the polarizer is rotated . Glan-Thompson prism.6 nm) . The extent of rotation can be measured by rotating the analyzer with respect to the polarizer till the rotation is fully compensated.. and 589. the monochromator has to be sophisticated. When the sample is introduced between the polarizer and analyzer prism.(ii) A monochromator (///) A polarizer (iv) An analyzer (v) Sample tubes (vt) A photomultiplier The light source can be a tungsten filament lamp. if a continuous light source i s desired. The Glan-Thompson prism is the best and used in most spectropolarimeters. optical rotation is produced. i. with either sodium vap or or mercury Vapor lamps. the polarizer and the analyzer are left perman ently crossed . Polarizer can be any of the three types: a Nicol prism. If tungsten fila ment lamp is used. The last is the least desirable as it never gives 100% polarization. For specific wavelengths xenon-or mercury-vapor lamps are used. a quartz-wave compensat or is introduced. each catering for a particular band-width. The trick here is to pass the plane-polarized light through a crystal . commercial instruments have just the once source from which L and R circularly light is generated. A rotati on of this plate from +45° to -45° produces respectively. The optical rotation produced is then compensated by a piece of quartz which rotates light in the opposite direction to that of the sample. d and l circularly polarized radiation. require two light sources: one for L and th e other R circularly polarized light. The two quartz pieces are laevo-and dextrorotatory. Instrumentation for Circular Dichroism Even an ordinary spectrophotometer can be adapted to measure circular dichroism. Recently composite CD/ORD measuring instruments have also become available and offer a wavelength range of 185-800 nm. The usual arrangement for this is to pass a plane polarized beam (obtained through a ny of the polarizers listed above for polarimeters) through a quarter wave plate. If a large wavelength region is to be covered.Other Optical Technkltes for Molecular Characterization 283 with respect to each other. The only necessity is to provide some means of producing d and I circularly polarize d radiation. in principle. Each would have a monochromator for wavelength sel ection.11 Depicts the schemat/c diagram of specialized CD instruments CD measurements would. several such quarter-wavelength plates would be required. The absorption by the sample of the r ight and the left circular components is then alternately measured to arrive at the CD spectrum. Source Lin(ar e polarizer Light of changing circular polarization Photomultiplier er Figure 9. Specialized instruments measuring circular dichroism are also available and have a wavelength range of 185-600 nm. In addition to this translational movement. The crystal is called an electroo ptic modulator a photoelastic modulator (the older CD instruments have a Pockels cell in place of this It has a remarkable property in that the polarity of the field determines which component will be transmitted. ROTATIONAL DIFFUSION We have seen that molecules in solution show translational movement caused by th e of the solvent. to its centre of mass. It has the units of reciprocal seconds and expresses . The output of photomultiplier is then processed to give the final result. The beam is then allowed to pass through the sample it is transmitted to the photomultiplier. each solute molecule . Recently composite CD/ORD measuring instruments have also become available and offer a wavelength range of 185-800 nm. The current is alternating and therefore the L an d R are transmitted alternately. CD instruments normally operate in the range of 185-600 nm. This motion is known as rotational diffusion and is descr ibed diffusion coefficient O.is subjected to an alternating electric field. Consequently its measurement assumes importance since it can provide an idea about the dimens ions of the solute particle. (1) Flow birefr/ngence Birefringence means double refract/on. All the three methods of determining 0 are discussed in the following pages. Fure 9. This is so since. w hich is a constant for a given solute. At this stage n o birefringence will occur. Solution of m/sotropic molecules . Such substances m/ght give rise to certain recognizable optical ch aracteristics when studied under polar/zing light microscope. its main use is in the study of dip ole moments. Its measurement is particularly valuable in case of molecules o f high axial ratio (length/width. el ectr/cal double refraction) or by means of hydrodynmIc shear (flow b/refrlngence. If however. stremng btrefr lngence.12). The y are (1) flow birefringence. for example long thin rods) where it can provide a precise measur e of the length of the solute particle in solution. can be expressed in terms of frictional coefficient e ' KTI frot Rotational diffusion coefficient is related to the dimensions of the solute part icle. on the other hand. double refraction of flow). Particles are randomly oriented. and (3) polarization of fluo rescence. btrefringence will be observed (F/gure 9. the molecu les in e solution are randomly oriented. at rest. Three methods are mainly used to determine rotational diffusion coefficient. Of these.ravels along the second s. Rotational diffusion coefficient. The double refraction can be given rise t o by the mIsotropy of molecular arrangement which causes I/ght to travel along one ax/s o f the crystal with a different velocity as compared to the velocity at which it t. (B} Hydrodynamic shear applied. (2) electric birefringence. The particles become aligned to .12(AJ Situation at rest. although electric birefringence can yield 0.Molecule 284 Biophysical Chemistry the average angle of rotation per unit time. the molecules are forced to zltgn themselves in a uniform way either by imposing an electrical field (electr/cal btrefringence. Many Crystalline substances are b/refringent since they possess two indices of r efraction along different es. does not give rise to b/refringence. This gives rise to a velocity gradient. FIw birefringence can be understood properly with the help of a commonplace exam ple. This effect is observed because of the difference in the amoun t of light reflected by the symmetrically oriented particles as compared to the randomly oriented one s. In a similar . A curious thing happens when an aged colloidal dispersion of vanadium pentoxide is stirred slowly.the stream line. (C) Microscpic situatin f a prlate ellipsoid particle in a velcity gradienL Arrows indicate stream line. the path of the stirring rod lights up since the colloidal particles ori ent themselves along the stream lines. Birefrlngence may occur in this state. The intensity of the birefrlngence depends on the flow rate and the shape of the particles. gives the rotary diffusion cons tant of the from which their size and shape are estimated by applying an appropriate Hydrodynamic shear : Flow birefringence is usually measured in an apparatus whic h concentric cylinders (Figure 9.13).Light source \ >Condensing lens C Y Diaphragm ). " Theoretical considerations indicate that the double refraction of the system app ears as a result of the equilibrium between the orienting effect of the applied field and the disorienting effect of the Brownian motion. For various reasons the oute r cylinder ------. the brightnes s of the field will be directly proportional to the birefrlngence. tlmt gives rise to flow birefringence. as a function of shearing force. In fact even Optically isotropic particles will give rise to birefringence provided that they have an anisotropy of the system as a whole and not the individual ansotropy i. The direction of the optical axis of the solution which is determined by the ave rage orientation macromolecules.Rotatin cylinder Other Optical Techniques for Molecular Characterization 285 way.t Condensing lens Po r . the fleld of polarizing microscope will light up due to birefringence. when a solution of nonspherical solute particles is subjected to hydrodynam ic shear. Two types of apparatus are available dependi ng whether the inner or the outer cylinder is rotated. but is independent of the birefrigence of the indivi dual particles. The light now falls into the lluld between the ¢on. space between the flxed and the rotating cylinders respectWe ly. o is the angular velocity of rotation.ntc cylinders aJter Imssing through a con denser and nlcol polarizer. G is gi ven by the relatWnshtp.Condensing lens (B) 9. (B) Cross-sectlon of the concentric cylinders. They are required only for measuring birefrirjence.Concentric cylinde Liquid between the cylinders ( Quarter wave plate Half-shadow wedge .flow birefrtnnee Olm. It is on these factors that the shear gradient G is dependent. Inddent lht is conjed by t. quarter wave plate and half-shadow are removed. Light now passe s through the analyzer nlcol and is then focussed by the condenser on to the observirj telesco pe through which optical patterns are observed. Rz and d are radius of the fixed cylinder. condenser on to the opening in the dW. R is the sum of Rz and d.phragm. For measurement of the extinction angle.11t AI Iommr representon oft/w. ¢oR2 Gd . The diaphragm now acts as lht sour ce. The optical system : The alignment of the molecules is determined by passing par allel light from a polarizing prism. The field appears totally dark. The higher the axial ratio of the particle (length/ width). Calculation of Rotational Diffusion Coefficient When the outer cylinder is not rotating. called the analyzer. in short. Anyone who has observed a small wooden stick floating on water would be able to visualize the alignment of the particles. the particles align themselves along stream lines and the solution becomes anisotropic and birefring ent. a quarter wavelength plate. when the solution is at rest. This velocity gradient produces a torque on the molecules tending to align their long axis tangential to the direction of the flow. a collim ator lens.e. The optical system for the measurement of birefringence consists of a light source. the greater the degree of alignment will be. however. when the outer cylinder is rotated. called the polarizer. The refractive index of this solution is no more the same in all directions. placed at right angles to the fir st. depending on their rotational diffusion coefficient). the rotating speed of which can be changed by voltage control. The outer cylinder is connected through a pulley system with a motor. turn very slowly. and an analyzer. and t hen through another polarizing prism. a polarizer. while the inner cylinder is stationary . The lig ht transmitted no . The macromolecular solution is placed in the space between the two concentric cylinders. remains linearly polariz ed when it comes out of the solution and is blocked by the analyzer which is placed at right angl es to the polarizer. through the solution.13). therefore. a filter. As the velocity gradient is increased. the particles align themselves in a manner which offers least resistance to the flow.286 Biophysical Chemistj rotating model is more preferable. The incident light coming through the polarizer. it is isotropic. the stick d oes not rotate but aligns its long axis in the direction of flow (the molecules. although its design is a bit complicated. However. As the outer cylinder rotates. Wherever the current of water is strong.. i. the angle between the long axis of the particle and the direction of flow (stream li nes) decreases. a velocity gradient is set up. The polarizer and analyzer are usually Nicol prisms or occa sionally polaroids. The sample sol ution enclosed between the two concentric cylinders is located between the polarizer and the qu arter wavelength plate (Figure 9. Th s ts the extinction angle 2. These four dark areas. The intersection of the long axis of the particle with the stream line also gives rise to the same angle. The same angle Is formed by the cross and the analyzer axis (A-A). It would be seen that light pa sses through every point except for four positions. . which correspond t o no birefringence. The cross forms an angle with the polarizer a xis (P-P).Particle P Figure 9.more remains linearly polarized. more light passes through the analyzer. th e former relationship utilized to measure .14). form a cross known as the cross ofisocltne (Figure 9. This dark cross is see n to form an P i/ \ A [ . However. but becomes elliptically polarized and passes t hrough the analyzer.14 The cross of tsocline. As the orientation of the particles is increased by increasing the spe ed of the outer cylinder. 14). Rotational diffusion coefficient can be calculated if G and are known. The relationshipbetween the rotary frictional coefficient and the shape of the p article is . At this point the parti cles for remain aligned in a preferred orientation. and is denoted by the symbol .(i The same angle.G kT Other Optical Techniques for Molecular Characterization 287 angle with analyzer axis and with the polarizer axis.n fact G depends upon the speed of t he rotating cylinder and the distance between the two concentric cylinders. is the Boltzmann constant and T i s the once rotary diffusion coefficient has been experimentally determined. This rotary frictJ. k. is made by the long axis of the particle with the stream line (Figure 9. This preferred orientation is denoted symbol a.on coefficient can b e calculated the following relationship where is the rotary frictional coefficient. The follo wing relationship is utilized for the purpose 60 tan 2X G After some time the rotating system attains equilibrium. can be calculated from it. The extinction angle depends upon the shear gradient G. X. the angle decreas es as the speed of the rotating cylinder increases. both the angles being of t he same extent. This angle is known as the extinction angle. I.al lengths can be calculated. a is a function of the dimensions of the solute particle and is descri bed by the relationship It is actually the rotary frictional coefficient from which the shape of the par ticle in of its axi. 15 The prolate ellipsoid model.axial length. . o is the viscosity of the solvent. The axial ratio (a/b) of the particle concern ed can be by intrinsic viscosity measurements.15) with semiaxes iand b where a> 5b 3 16 moa 9. For a long prolate ellipsoid (Figure 9. The model is obtained by rotation of an ell ipse of major axis a and minor ax/s b about the a axis. The Values of X obtained are then plotted against (Figure 9. is measured for of G by varying the cylinder is rotated.16).= dG 12 @ " Rotational diffusion can be measured by another Here.16 to G by dz . When a is less than the relationship is as follows I G = 4 120 It is thus possible to relate initial slope of the plot shown in 9.288 32 ob3 3 180S Biophysical Chemistry For oblate ellipsoids Here b is longer than Figure 9.16 A schematle representation of the plot to determine Q. . c is measured for different values of G and dz dX I dG is related to 0 by -. tropomyosin . be calculated by the equation where . Relationship Between Flow Birefringence and Molecular Weight If is the angle of rotation of the analyzer in degrees. Dimensions of several macromolecules have been determined using flow For example. is the wavelength of the incident light in vacuum and S is the path solution. olecules by the following equation An = BnM C . Once the degree of birefringence has been calculated. each time the cylinder. Both these values are in agreement with values for t he macromolecules found by other hydrodynamic methods.rotating in opposite directions. The differenc e between two positions is equal to 2). it can be related to the w eight average molecular weight of the macrom. btrefringence of the sol ution. the length of the major axes of flbrinogen and serum albumin were f ound to 670/ and 190/ respectively. Some other example whose shape has been determined using flow birefringence are myosin. the angle between the polarizer axis and the cross two times.I dG 120 (Experimentally. tobacco mosaic virus etc. 17) (2 e provide two different informations about the Rir-e "macromolecule. The fiel is usually applied as a short pulse of the order of microseconds. In flow blrefringence macromolecules were aligned with the help of hydrodynamic shear. These serious drawbacks have limited the application of this method for molecular weight determination. it can be of uti lity only when in which the macromolecules are placed is relatively nonconducting. the method depends upon assumptions regarding the mole cular geometry which involves an appreciable degree of uncertainty. The optical system is so desigrted that whe n the molecules align and the solution becomes birefringent. The degree of alignment i s then determined i from the birefringence produced. Inspite of providing a considerable amount of information about macromolecules. Although fl0w birefringence has been used many times for determination of molecu lar weight of macromolecules. Macromolecules are forced to align by application of electric field. Moreover it requir es evaluation of empirical constants. The measurement involves determination of int ensity of light passing through crossed polarizing lenses and falling on a photomultiplier tube where it is converted to an oscilloscope signal.Other Optical Techniques for Molecular Characterization 289 where Bn is a constant characteristic of the solute. C is the concentration of t he solution and M is the molecular weight. a tracing immediately appears on th e oscilloscope screen. The rise and decay of birefringence (Figure 9. Electric birefringence Another technique to study the dimensions of macromolecules on the basis of thei r charge properties is electric birefringence. 2. the method has a serious disadvantage which limits its application.properties of t he molecules. At the onset of the electric pulse. the . In electric birefringence an electric field is used to align the macromolecules and the alignment is determined by the electrical. Unlike other hydrodynamic methods. provides a 9. activation of a protein in whic h are cleaved from the protein can be studied with the help of electric birefringe nce.. Polarization of fluorescence Polarization of fluorescence has been often used to determine rotational diffusi on coefficients ' macromolecules. The decay of . From the dipole moments one can determine the pattern of distribution of charged groups on the . activation (where two peptides with a charge of-4 are cleaved by the action of t he thrombin) can be studied with the help of this technique. The method is of considerable utility to study reactions in which charge take place on the protein molecule. the technique is utilized to study of a charged hapten to an antibody specific for it. this technique is not limit ed to . pH 7. For example.9) about the shape of the macromolecule. then.17 Oscilloscope tracing showing the precise measure of the rotational diffusion coefficient. Time ( sec) birefringence is due to rotational diffusion coefficient of the macromolecule. I M which as we have seen earlier can provide an idea glyclne.shape of the rise of birefringence provides information cay about the orientation of the induced and permanent dipole moments of the molecule. For example. trans/ent e/ectrlc blrefr/ngence of flbrtnogen in solution (0. charge protein can also be studied. The decay. In a likewise manner. In practise this is done by measur ement of fluorescence polarization as a function of viscosity and subsequent extrapolatio n of the data to infinite viscosity. seldom takes place. Those molecules whose electric dipoles are in closest alignment with the field direction will absorb the most. On the other hand. The rotational diffusion coefficient of these chromophores ( and therefore the macromolecules t o which they are bound) can then be calculated by comparison of the resultant polarization wi th that which would be obtained if no diffusion took place. Moreover. In solutions. When the exciting beam used is polarized. If the alignment of the solute m olecules does not change within this time. In this case a maximum value fo r the fluorescence polarization will be obtained. Brownian motion leads to rotational diffusion which randomizes the orientations of chromophores during this period (10 nsec). however. the method is free from those limita tions suffered by hydrodynamic methods and is sensitive to very small quantities of sample. This is the simplest method by which rotational diffusion co efficients of macromolecules can be determined. Fluorescence polarization may be characterized by emission anisotropy. Thus with these ch romophores only a partial depolariztion can occur during lifetime of the excited state (10 nsec) . The lifetime of excited state of a solute molecule after a bsorption of llght is usually 'of the order of 10 nanoseconds (nsec). This. those chromoph ores which are tightly bound to macromolecules have a slower rate of rotational diffusion ( characteristic times for this process are of the order of 100 to 1000 nsec). The absorption of this light now depends on the orientation of the electric dipo le of the solute molecules. the emission anisotropy is given by the following expres sion r+ 2I± . Brownian motion has a large effect on small molecules (faster rate of rotational diffusion) whose alignment gets randomized fast and t heir solution shows complete depolarization of fluorescence. This results in fluorescence depolarization additional to that caused by initial random distribution of chromophores.290 Biophysical Chemistry In this technique the sample is irradiated with polarized light of the w avelength of absorption. the resultant fluorescence will show the same extent of polarization as that represented by the absorption distribution. the intensity of the scattered light can then b e expressed as a . As per this theory the incident electroma gnetic impulse of a beam of light causes electrons of an isotropic particle to vibrate in unison w ith it. Lord Rayleigh formulated the fundamental laws of light sca ttering in 1871 by calclflating the polarizability of individual gaseous molecules placed in an oscillating electromagnetic field of a light beam. LIGHT SCATTERING If a parallel beam of white light illuminates a colloidal solution. The oscillating electrons now b ecome sources of scattered or diffracted light which will mostly have the same frequency as th at of the incident beam. it is observable in all transparent media like gas.where Ii and I. This phenomenon. is commo nly known as the Tyndall effect after its discoverer J. Although light scattering is more pronounced in colloidal solutions. Normally the two polarized compon ents are measured using the same photomultiplier. Tyndall. The intensity of the transmitted beam is consequently lowered. Il and I± are in practice determined by measuring fluorescence intensities at righ t angles to the exciting beam. caused by the scattering of light. faint bluish light can be observed laterally. The intensi ty of the transmitted beam. and therefore. This is made possible by using a rotating polarizeralterna Uvely transmitting the two directions of vibration (Figure 9. An oscillating electric moment is thus induced in the particle. any pure liquid solution and even in crystals. are the principal components of the polarized fluorescence vibra ting in parallel and perpendicular directions respectively (with respect to the direction of the exciting beam).17). particles or molecules) p er unit volume. = turbidity. and Putzeys and Borsteaux of France were the first to apply light scatt ering toward the calculation of molecular weight in 1935.is the flxd polarizer. Pz .where Other Optical Techniques for Molecular Characterization 291 function of the number of centres of scattering (i. P2 is the rotating polarlzer which alternately transmits Ill and I .e. A mathematical relationship describing light scattering phenomenon can be writte n in much the same way as the Lambert's law I Io = intensity of the incident light. D is the photomultlpler. Thus. Smimov and Bazenov of U. if a value for Avogadro's number is adopted.S.. depends upon the number and size of the scattering particles..18 A simple setup for Jluorescence polarization measurement.R. vf is the Jluorescence frequency. I = intensity of the transmitted light. we is the exciting frequency. and x = the path length. Turbidity. the turbidity can be expre ssed by the . P we y " p Ill I Figure 9.S. quantitative measurement of l ight scattering can provide an idea of the molecular weight of the scattering specie. S is the Ikjht source. For li ght scattered at 90° to the incident beam in ideal systems. Monochromator s and collimators are not shown. .following mathematical relationship 32z 3 X4 a where n = index of refraction of the solution. a is replaced by its equivalent CN/M (where C is the concentration of the solute in g ins per ml. = the wavelength of light in vacuum. To introduce a term for molecular weight into the above mathematical relationshi p. no = index of refraction of the s olvent. and a = the number of solute particles per ml. M is . Lens. and N is Avogadro's number). The presence of any of the two Impurities stated above will lead to false scattering values. As it is. usually a photomultiplier which records the scattered r adiation.292 Biophysical Chemistry the molecular weight. Fine pore filtration or high speed centrffugation might be performed to get rid of the dust particles. Presence of aggregates must also be avoided at all costs. in molecular weight determinatio n if formation of aggregates is not guarded against. Molecular weights obtained by light scattering data are weight average molecular weights (See Chapter 4). The solutions must essentially be dust free. This equation can be used to calculate molecular weight after obtaining light scattering data. Figure 9. D is the detector..apparatus for light scattering nts. Monochr omatic light is obtained from a laser. D is mounted on .source. For nonideal systems the relatio nship takes the following form . The mathematical expression c an now be written as where k is a proportionality constant defined by the relationship The relationship /C = kill is for ideal system. kC 1 =m+2RC M where R is the interaction constant and is dependent upon the solvent used. The apparatus used for measurement of light scattering is called light scatterin g photometer. The method has a srious drawback in that it is very sensitive t o the presence of aggregates and large scale errors may creep. L focusses thecollimated beamofwavelength and intensiJloon to thescattering cell.19 Schematic of a typical.19 is a schematic representatibn of a typical light-scattering experime nt. [ ser t OO Figure 9. molecular weights obtained by li ght scattering are considerably higher than those obtained by other methods. . owing to th e rapidity of the method. Although in the present text we have touched only the aspect of molecular weight determination by light scattering measurement. scattered intensity at any angle 8 can be measured. Moreover. light scattering offers unique possibilities of following the kineti cs of macromolecular reactions involving a change in the size or shape of the dissolved particles. it should be noted that the pheno menon is also useful in providing data on the size and shape of the dissolved macromolecu les as well as information on the thermodynamic properties of the system.a moveable support so that by chancjing its position around the scattering cell. A wave fro nt. The importance of this method of s. the beam PM'will be reflect ed partly at M' along RUN'. interference will . W. if the two beams are out of phase. 10-s cm as the distance b etween individual atoms in a crystal. 9. A pattern of this type is generally referred to as the Laue pattern. and partly at Q on the second plane along QMN. Bragg were the first to call at tention to the fact that. Part of the beam.. W. the distance Plt QM as compared to the distance LM. The wavelengths of X-rays are of the same order. parallel and equidistant lattice pl anes. LP.20. LM. at the angle of reflection. viz.Other Optical Techniques for Molecular Characterization 293 X-RAY DIFFRACTION It is very instructive to wonder as to where molecular biochemistry would be tod ay if there had been no X-ray analytical methods to discover the atomic architecture of biop olymers such as proteins and nucleic acids. and W. CC. will reinforce each other and a maximu m intensity beam will result. Knipping verified this idea by passing a beam of X-rays through a crystal of zinc blende. BB. of X-rays is approaching a series of identical. Similarly. the crys tal playing the part of a diffraction grating. Previously this method was used to calculate the wavelengths of X-rays. This'similarity led Max yon Laue in 1912 to make a brilliant suggestion that crystals could act as natural and very fine three dimensional di ffraction gratings for X-rays.. To understand the theory of this method better.tructural anal ysis can be stated simply by saying that the structure of DNA was discovered largely due to the availability of X-ray crystallography data. This resulted in a deflrdte diffraction pattern on the exposed photographic plate. If the length of the path LMN differs from that of PM" QMN by a whole number of wavelengths. Friedrich and P. the se cond beam travels a longer distance than the first.L. An X-ray bea m striking these atomic planes will be diffracted in such a manner as to cause either interferenc e or reinforcement of the beam diffracted from the outer plane. The whole beam would behave as if i t has been reflected from the outer surface of the crystal. will be reflected at M along RUN. since a crystal is composed of a series of equally spaced atomic planes.H. AA. then the two beams will be in phase at-M. consider Figure 9. i t may be employed not only as a transmission grating. To emerge along RUN. However. which constitute the atomic planes of the crystal. but also as reflection grating. and is known as the order of reflection..4. and the intensity of the reflected beam will be less than maximum. it becomes clear that .2.3. It is thus clear that the condition for maximum intensity beam is that the distance PQM-LM = n where is the wavelength of the X-ray used and n is an integer equal to 1. N R Iure .result. etc. and draw a nother from M to Rerpendicular to AA.0 ReJleetlon of X-foxes y l:u"lle[ loes If we now draw a perpendicular from M to the extension of line PM" Q. BB and CC. Mor eover.PS = SR Thus. Thus PQM = PR But LM -. the distance between the successive lattice s of the crystal. since MS was drawn p erpendicular toPR.PS Biophysical Chemistry Therefore. . The relationship allows calculation of the ratio )Jd by measuring n. PQM = LM = PR . is applicable to reflection from other planes parallel to AA and BB. This simple expression directly relates the wavele ngth and order of reflection of theX-rays to the interplanar distance d and the angle of maximu m reflection. Therefore rvX = 2d sin (. 0. the wavelength. X-ray diffraction can thus tell us a great deal about the structure of the crystal. Now. Z. can be calculated. if d is known. from the figure it is clear that QM = QR. known as the Bragg equation. Z. if t he wavelength. SR = rv From the figure it is also clear that angle SMR is (3. d.294 But. This relationship. of the X-ray Can be calculated. and (3. More importantly. SR Sin(3 MR and SR = MR sin 0 = 2d sin (3 where d is taken as the distance between any two atomic planes in the crystal. of the X-ray being used is known. Th us. The diffracUon pattern of a crystal can thus be compared to a 'fingerprint' and prov ides identities of the components of the crystal. if one considers the direction and the intensity of the diffracted beams.Every atom in a crystal scatters an X-ray beam incident upon it in all direction s. but with less than maximum effe ct.the position of the diffracted beams depend only upon the size and shape of the repetitive unit of a crystal and the wavelength of the incident X-ray beam. The orientation of the plane would then be described by the spatial orientation of this line. are made up of a very large number of atoms. even the smallest one. W hile maximum contribution to the intensity of the diffracted beam is afforded by the atoms lo cated exactly on the crystal planes. As all crystals. The Bragg eq uation described above states the condition for diffraction of a beam of X-rays from a crystal. although . Th e length of the . maximum destructive interference is meted out by the atoms e xactly halfway between the planes. th e chance that these scattered waves would constructively reinforce would be almost zero but fo r the fact that the atoms in a crystal are arranged in a regular repetitive manner. Therefore. the intensity of the diffracted beams depend also upon the type of atoms in the crystal and the location of the atoms in the unit cell (the fundamental repetitive unit). A plane may be represented by a line drawn normal to the plane. Reciprocal Lattice Concept Diffraction phenomenon can be interpreted most conveniently with the aid of reci procal lattice concept. no two substances can have absolutely identical diffractio n patterns. Atoms located at intermediate positions interfere constructi vely or destructively depending on their exact location. The scattering power of an atom increases as the number of electrons it possesses increases. near the origin. y '. namely. 203 003. Norrnals t o the planes are also shown.Other Optical Techniques for Molecular Characterization 295 normal is usually fixed in an inverse proportion to the interplanar spacing of t he plane it represents. since the distance of each point from the origin is an inverse or reciprocal of the interplanar spacings. The normals to these planes ar e called the reciprocal lattice vectors ha and are defined by The lattice array is defined by three reciprocal lattice vectors in three dimens ions. [Y. '302 "- / ' I02 301 (00} " (0| Figure 9.21 shows.21 A profde of several planes in the unit cell of a crystal. this is called a reciprocal /att/ce. When normal are drawn to all the planes in a crystal from a common origin. (101L and (102} planes. the (I00}. the traces of several planes in a u nit cell of a crystal. Figure 9. the t erminal points of these normals constitute a lattice array. (001}. The magnitudes of these three vectors are given by b =oo = d 010 The directions of these vectors are defined by three interaxial angles a'. However. . with the other parameters described above. while the crystal plane represents the diffracted beam's directi on.22(A) can also be interpreted as shown in Figure 9. The li ne . "l -le construction in Figure 9. ABC represents the direction of the incident X-ray beam. The llne BD forming an axgle 20. In the above expression the numerator represents one side of a right triangle wi th 0 as another angle and the denominator represents the hypotenuse (Figure 9.22(A)).296 The Bragg equation now might be rewritten in a form that will relate the glancin g angle O. .e diamete r of the circle. Then the .AD. drawn through the origin of the circle and making an angle 0 with the incident beam defines a crys tal plane which satisfies Bragg diffraction condition.22(B}. with the incident beam and an angle 0. The vector h originates at the point on the circle where the dire ct beam leaves the circle.for Molecular Characterization line CD is the reciprocal lattice vector to the reciprocal lattice point Dh lyin g on the circumference of the circle. the distance d between successive lattice planes of a crystal can be calculated. Two methods are most popular. Now when a reciprocal lattice point lies on the "sphere of reflection" ( a sphere formed by rotating the circle upon its diameter -'-). Bragg equation makes it clear that if the glancing angles 0 are measured f or the various orders of maximum reflection. Diffracted / beam beam A Figure 9. the Bragg equation is satisfied. The rotat/ng crystal method and the powder method. It thus follows that with rotation of the crystal. Incident X-ray beam Sphere of reflection . a reflection will emanate from the crystal at the sphere's centre and will pass th rough the intersecting reciprocal lattice point. Und er no other conditions will Bragg equation be satisfied. and if the wavelength of the impinging X-r ay is known.22 Diagrammatic representation of the diffraction condition In a diffraction experiment the crystal can now be pictured to be at the centre of a sphere of unit radius. . The Determination of Crystal Structure "the.23). Whenever a reciprocal lattice point will in tersect the sphere. The reciprocal lattice of this crystal will then be centred at a point where the direct beam leaves the sphere (Figure 9.297 Other Optical Techniques . the reciprocal lattice will also rotate. 1 .........°ooI . |/'-.. 1 '2 '-' 4 - Crystal rotation axis ..... 3 ÷------2 . In order to determine the positions of maximum reflection intensity. The position of the crystal at any given time can be read on the scale L. This method necessitates the use of a large crystal with well defined faces. 8.24) or a photographic film. which satisfy the Bragg equation. since reflections of several orders must be examined for a large number of faces. A narrow 0 Figure 9. The beam then falls upon a known face of crystal C. X. the experimental set up is such that a monochromatic X-radiation is Incident upon a crystal which is rotated about one of its axis.298 Bophgscel ChemLstn3 Rotating crystal method : As the name suggests. beam of X-rays falls on the finely powdered substance P.25 Diagrammatic repre sentation of the principle of the powder method. and the Intensity of such a ray is determined. The rays reflected by the crystal are allowed to reach an ionizing detector D through another sllt. The Imwcler mthod : In 1916.Hull devised the powd er method of X-ray crystallography which went on to become a widely used method. This.W. The procedure is repeated for all the planes of the crystal. however. give the strongest reflections. P. lgure 9. The powder is usually coated on a hair or inside a .either of the Bragg X-ray spectrometer (Figure 9. P. The apparatus is so designed that the reflected ray always enters the detector D. use can be made. The detector is usually a Geiger cbunter. Moreover. The glancing angles. the total labor Inv olved is enormous.24 Diagrammatic representation of Bragg X-ray spectrometer. Description in the text. Scherrer and A. mounted on a rotating turn table. In the X-ray spectrometer a beam of X-ray of a definite wavelength cming from the anticathode of an X-ray tube. is compensated by relatively simple Interpretation of the results . Debye. is allowed to escape through a slit S. Description in the text Thereflected beams lie as spots on the surface of cones which are coaxial with the rotation axis. As crystals In the fine powder are oriented in all directions. Circular cones will be produced In a similar manner for other planes. for a particular reflection. Chol of X-Radiation Bragg's equation can be rearranged and written as follows: . I. Thus. The diffracted rays collide with the photographic film F which is arranged In a circular arc with P at its centre. a large number will have their lattice planes In the correct positions for maximum X-ray reflection to occur.25). and the spot of Intersection of undiffracted beam with film. All crystalline particles whose (100) planes are In proper angle with the Incident X-ray beam will give rise to first order maxima In directions lying on a circular cone (Figure 9. the only necessity is to measure the distances such as/D or IE (Figure 9.25).thin walled glass tube. In order to measure 0. The distance between the powdered substances P. Other crystals whose (I 10) planes are in proper angle with the Incident X-ray beam will produce another circular cone. The st ructure of the crystalline material can then be determined. is fixed for a given X-ray camera system. and (B) Powdered sample. it is nece ssary to mount Crystal Shellac Powdered sample Glass capillary Picein wax Glue F@ure 9.Brass Other Optical Techniques for Molecular Characterization 299 The resultant of the terms in the parenthesis never exceeds unity.26). This radiation darkens the film making it difficult to locate the di ffraction maxima sought. is fastened to a brass pin (Figure 9.' On the other hand. In such a case the crystal absorbs the radiation and emits fluorescent radiation in all directions.26 TWo methods of specnen mounts for X-ray di. For protein crystals. in case of a crystal having a very large unit cell.'actloru (A) Single crysta l. Clearly then. The crystal in this case should be of such a size as is co mpletely bathed by the incident radiation. Thus. Due to this u se of long wavelength radiation is not very useful as it limits the number of reflectlons t hat can be observed. usage of short wavelength radiation results in a crowding of individual reflections making the interpretat ion a tough job. however. the wavelength of the chosen radiation should be outside the absor ption range of the crystal material. Specimen Preparation Use of single crystal is preferred for structure determination because the data so obtained is easy to interpret. the chosen radiation should not fall Within the absorption range o f the crystal material. the crystals in an enclosed space. The crystal is generally affixed to a glass capillary wh ich. in turn. Additionally. This is so since the protein crystals contain . ex tremes. the choice of radiation has to be a compromise between the two. When a single crystal of sufllcient size is not available. If such crystals are exposed to air. liquid evaporates and the cr ystals shrink deteriorating the quality of the X-ray pattern obtained.26). At other times the powdered substance might be coated on to a hair and the assembly is fixed to a brass pin (Figure 9. protein cry stals are placed in [ thin wall capillary wliich is then sealed at both ends. Sometimes the interior of the capillary is coated with a hydrophobic film to make for a cleaner mount. Prom the space group a nd the unit cell volume. a polycrystalline agg regate is formed into a cylinder whose diameter is smaller than the diameter of the incident X-ra y beam. Therefore.within themselves a large amount of solvent from which they are grown.. If we now denote the molecular weight of the a symmetric . About half the volume of pr otein crystals is of solvent. the volume of the symmetric unit can be calculated. X-ray Diffraction and Molecular Weight A unit cell might contain more than one asymmetric units. the number of molecules in the symmetric unit can be calculated. If the app roximate molecular (M) of the macromolecule is known from other methods. There are. New York. and Beychok. S.J.) Part C. 6.J. Edsall. in Physical PHnciples and Techniques of Pro teln Chemistry. 446. Imahori. The molecular weight as determined by X-ray data is therefore termed as "crystal molecular weight". be borne in mind that the molecular weight as determined from X-ray diffraction data of the crystal mi ght be a multiple or submultiple of the true molecular weight and might also differ from the value of the molecular weight determined by other methods. E. Sears.113. (R. 7.. 4. 27 : 6 75. Shape and. K. N. Morris. p.. in New Techniques in Biophysics and Ce ll Biology. in Physical Prlnctples and Techniques of P roten Chemistry. New York. Suggestions for Further Reading I. Academic Press. . Academic Press.T. p. and Sanderson. G.. J. and the number of asym metric units per unit ceil by n.H. Methods in Enzymology.. the volume of the dry unit cell by V. 5. It should however. Beychok... pp. the following expression Can be written where k is a constant.J.W. one can arrive at a fairly accurate value of the molecular weigh t of the molecule. Pain. Leach.R.)p. Vol 1. (S. The Sze. Greenfield. 358. 154 : 1 288 (1966). and Nicola. 1973. ed.1 V 300 Biophysical Chemistry macromolecule by W. G. Neurath and K. D.Hydration of Protein Molecules in the P roteins. Adler.A. John Wiley. in fact. 1973. A. eds). Leach. Vol.J.D. 2.J. few other methods of molecular weight determination which wi ll give a value as accurate as this from a single simple experiment.. 549-716. Part B. ed. Academic Press. CircularDichroism of Biological Macromolecules. London. New York. 1973. Barley. Smith. (H. and B. eds.) Part C. 1973. Science.R. From the above equation it is obvious that if one knows the number of asymmetric units in a unit cell.-S. 3. 1953. and Fasman. (S. N.l. J.A. eds.L.. The Theory of Optical Activity. New York. New York. CB$. Dickerson. (S. . John Wiley. Stacey. Yoshioka. 1970.. London. & Settle. S. New York.. Neurath.. New Delhi.J.N. 1956. Vol. 233-287. ed. 13. F. R. H. Dean. 8th ed. K. New Yor k. I0.H. Instrumental Method of AnoB.. J.) Acadera/c Press. Dielectric Properties of Protelns II. Meritt.. Willard. Part B. X-ray Analysis and Protein Structure in The .Caldwell.E.] Academic Press. 2 nd ed. H. ed. Wfley-Int erscience. (S idney Leach.. Jr. K. D. (R.H. Leach. 8. ed. and Tbwnend.. L/ght Scatter/ng in Physlca/Chemistry. Feeney. H. and Eyring. 1988. I I. Ele ctric Brefrlngence and Dichrolsm in Physical Principles and Techniques of Protein Chem/stry. Pain and B. Tlmasheff.} Academic Press. Smith. in New Techniques in Biophysics & Cell Biology.. New York.).A.. and Watanabe. 12. Part A. Fluorescence Nanosecond Pulse Fluorimetry. L. pp.A. Vol. (H. R. /. 1975.J. 1964.J. 1971. 1969. 2.jht Scatterb in Phy$1ca/Pr/nc/es and Tec hniques of Protein Chemistry.ProteUs. Academic Press. 9. 2. This. The particles in the solution will t hen a centrifugal force is acting upon them in addition to the gravi tational Rwas in 1923 that Svedberg and Nicols employed a centrifuge for the first time t o increase so as to speed up the rate of sedimentation for the purpose of measuring sizes. The vessel and the rotor Iki are also shown. only on standing for extended period of time will concentration gradients develop in undisturbed solu tions. the in a vessel rotating at high speed. A yplml rotor with holes to accomodate vessels Jled wh suspension.force of gravity. A solution to this difficulty is to increase the gravitational potential energy by . and molecules to the study of molecular weights of macromolecules. Thus.surrounding medium far outweighs the directing. This is because . for small gravitational force is so minute that the random bombardment of the molecules of . All other parts that different centrifuges consist of are accessories for maintaining the environment within which the centrifuge operates or for modifying the working of the rotor itself during emergencies. This pioneering work was followed by gradual development of ultracentrifu gation is probably the technique most responsible for our current understanding of cell ular The applications of this technique range from collection and separation of cells .CENTRIFUGATION Maeromolecules are almost insensitive tO gravitational settling. sedimentation would then appear to be completely useless for separation or of macromolecules in solution. however. is not completely true. BASIC PRINCIPI. OF SEDIMENTATION An object moving in a circle at a steady angular velocity will eperience a force . The basic components of a centrifuge are (t) a metal rotor with holes in it to accommodate a vessel of liquid (Figure 10 I{A} and (tO a motor or alternative means of spinning the rotor at a selected speed. to cent/meters. o).. directed This is the basis of centrliation. F.. RCF. and the radi us of r. The referred to as the relative centrifugal force.. Angular velocity in radians. F= ¢' r . RCF is more frequently refer red to 'number times . (I} F might be expressed in terms of earth's gravitat/onal force if it is divided by 980. collect/rely determine the magnitude of the force F. centrifuges owing to their designs would have different radii (distances between the axls and the.:'.I19 x 10-5) (12. it is better to express the acting upon a particle in terms of RCF rather than the rpm. 109" '00 .000) 8. A nomogram based.303 The speed of a centrifuge rotor might be expressed in terms of rlSm or RCF. (ii) RCF = {I.8 and 8. SOoOOO 40. 4.8 = 7.734 x g.00 15O0 15.0 cms.891 x g..0 = 12.119 x 10-5) (12.2. In view of the above. Consider two centrifuges operating at the same rpm. 12000. Radius Relative Rotor speed (in cm) centrifugal force (rev rain-') 20151000 00. on (4) relating RCF to rotor speed and r is given in Figure 10.000)2 zt. have different radii. Calculating for the RCFs both the centrifuges we get (/) RCF = (1.00O . however. and to avoid ambiguity in scientific work. two centrifuges. middle of the sample tube) which would give rise to different RCFs at the is illustrated below. Howe ver. it have to displace some amount of the medium of suspension. one can.2 Relationship between radius. the rate of sedimentation of a given particle would also depend upon its characteristics such as its density.find out the value of the relative centrifugal f orce.I000 50.will also tend to affect the rate of of the given particle. Apart from RCF. By aligninj the values of the radius and rotor speed. This displacement will result .310 ". Consider that centrifugal force is being applied to a particle. viscosity -. The characteristics of the medium in the particle is suspended -. As he particle s ediments. and its radius. The right side and the left side of the RCF scale correspond to the right and left side of the rotor sp eed scale respectively.its density. 5000 10. rotor speed and relative centrifugal force. This can be achieved by two methods.(I0) Centrlfugatlon 305 4. The above equation makes it clear that a mixture of heterogeneous particles ( fo r example. This is the principle of the so called dtfferent' centrl fugatton and we will discuss it later. broken cells) can be separated by centrifugation on the basis of their densities and/or size.. We can repeat the procedure with the supernatant and pelle t different organelles differently.. 0rganelles differ in their size as well as their densities. 9 t=-. Natural ly. we can removethe supernatant (the suspension above the pellet) and resuspend the pe lleted particles in a suitable medium. the slower will be the particle movem ent. Once a particle reaches the bottom and packs there (such a packed deposit under sedimentation is called a pellet). . The higher the viscosity of the medium.x Inr--b where t = sedimentation time in seconds. An expression for sedimentation time can be obtained by integrating equation (8}.. Letting the particles (different organdies in case of broken ceils) sediment to the bottom of the tube. rt = radial distance from the centre of rotation to the liquid meniscus. Letting the particle be sedimented for a fixed time at a fixed sedimentation vel ocity. and rb = radial distance from the centre of rotation to the bottom of the tu be. A different way to look at the above mathematical expression is to consider the time required for a spherical particle to sediment to a given distance in a centrifug al field as a function of the variables listed above. they will sediment at different rates and thus some will reach the bottom before the others. the tubes have to be placed diametrically opposite to each other to disp erse the load evenly. In all these centrifuges the rotors are mounted on a r igid shaft. . Larger capaci ty (4-6 dm) centrifuges are also available and there are centrifuges whose rotors can accomm odate bottles of fairly large capacity.Since each particle sediments at a different rate. "Desk Top Centrifuges These are very simple and small (can be placed atop a desk and hence the name) a nd are the least expensive. Separation can be carried out in 10.clinical centrifuges since most of the clinical work is done by these models. T heir maximum speed is usually 3000 rpm and they do not have any temperature regulatory system . It is therefore very important that the contents of the centrifuge tubes are balanced accurately and that they are never loaded with an odd number of tubes. We defer the theoretical discussion here to certain other sections where it will be more useful. These are also known as . 50 or 100 cm tubes. all the particles will be fou nd at different zones within the tube ailer sufficient time has been allowed to pass. However. INSTRUMENTATION Many centrifuges of different designs are available in the market. all centrifuges can be roughly categorized into three different types on the basis of their oper ating speed. if the rotor i s only partially loaded. Moreover. This principle i s exploited in density gradient centrlfugation which. yeast cells or bulky precipitates of chemical reactions. These precautions are true for all types of centrifuges discussed here. They are normally used to collect rapidly sedimentlng subst ances such as red blood cells. again will be discussed later. The main difference between the centrifuges of this category is the maximum carr ying capacity. Apart from this the ultracentrifuge has a refrigeration system which ca n maintain the temperature of the rotor between 0° and 4°C.306 Biophysical Chemistry High Speed Centrifuges High speed centrifuges can operate with maximum speed of up to 25. The drive shaft on which the rotor is m ounted is merely 1/16 inches in diameter.000 rpm providing centrifugal force in excess of 500. Neither are they useful in se dimenting individual molecules for want of sufficient centrifugal force. The Ultracentrifuge The ultracentrifuge can operate at speeds up to 75. cell debris. they are of little use in isola ting smaller organelles such as the ribosomes. They are usually equipped with refrigeration equipment to remove heat generated due to friction between the air and the spinn ing rotor. The temperature can easily be maintained in the range 0-4°C by means of a thermocouple . The sh aft is made up of aluminium or titanium alloy of high tensile strength to withstand the grea t forces generated during centrifugation. all centrifuges possess an overspeed device. These instruments are routinely used t ocollect microorganisms. microsomes. large cellular organelles. precipitates of chemical reactions and immunoprecipitates.5 dm3. Although these centrifuges are useful in isolating sub-c ellular organelles such as the nuclei. To eliminate this source of heating.000 g centrifugal force in the process. etc. The resolution power of an ultracentrifuge can be gauged from the fact that it c an resolve two types of DNA molecules differing only in the fact that one of the two types contains SN (the . The small diameter allows the shaft to flex duri ng rotation accommodating a certain degree of rotor imbalance without spindle damage. mitochondria.000 g. To contain such explosions the rotor chamber is always enclosed in a heavy armor plate. At such speed the friction between air and the spinning rot or generates significant amount of heat. cells. To prevent the rotor from operating at speeds which exceed its maximum rated spe ed. Operation of rotor at excessive speeds can result in an explosion with the rotor being torn apart. the rotor chamb er is sealed and evacuated by two pumps working in tandem making it possible to attain and hold v acuums of 1 to 2 . The highest carrying capacity may be 1.. lysosomes etc.000 rpm provid ing about 90. Moreover.g. while that carried out with a desire for characterization is kno wn as ana/yt/cal centrifugation (we will discuss both these types in the subsequent pages). Over the years the ultra has been used to isolate viruses in pure fo rm. The ultracentrifuges also are of two types -. but to some extent their confo rmations also. RNA. obtaining a sub-cellular organelle without any contam ination from other organelles or fractionation of a macromolecule) or with a purpose to chara cterize a macromolecule or sub-cellular particle with respect to its molecular weight or s edimentation coefficient..the preparatory ultracentrifuge. the ultra has also been of much use in such analytical applications as the characterization of macromolecules (proteins . This has allowed careful analysis of their composition. Ultracentrffugation can be carried out either with a desire to obtain certain bi ological material in isolation from the components that it associates with (e. Centrifugation for isolation and purification of components is know n as preparatory centrifugation. isolati ng a cell type away from all other cell types. it also Is . Wit h this backdrop it is easier to understand how the ultracentrifuge has helped biochemists in partic ular and the biologists in general to not only isolate. a nd the analytical ultracentrifuge.heavier isotope) in place of the naturally occurring 4N isotope of nitrogen. All the description given above was that for prepara tory ultracentrifuge. Given below is a description of the analytical ultracentrifuge. Analytical Ultracentrifuge The analytical ultracentrifuge is more or less like the preparative ultracentrif uge described above in that it operates at almost the same maximum speed providing the same RC F. DNA) not only with respect to their molecular weights. but to study the structure-function r elationship of those subcellular organelles which could previously be only observed under an el ectron microscope. the alloy used in the rotor and the s haft are the same. The optics used in a n analytical centrifuge are either Schlieren optics or Rayleigh interference optics. It. The main function of the counterpoise is to counterbalance the analytical cell. The rotor of an ultracentrlge is elliptical (Figure 10. The upper and lower planes of the analytical cell are transparent having quartz or synthetic sapphire windows. It has a ca pacity to accommodate about 1 cma of sample. One cell is known as the analytical cell while the other is known as the counterbalance or the counterpoise cell. The windows are provide d for the passage of light to monitor the progress of centrifugation. The rotor holds these cells vertically whether it is at r est or rotating. differs from the preparative ultracentrifuge in having differ ent type of rotor and in possessing a specialized optical system to monitor the progress of centrf fugation. however. however.3 A) and has two holes for ho lding two centrifuge cells. The analytical cell is sector shaped and can hold a liquid column about 14 mm high. It i s a precisionmachined block of metal with two holes drilled at calibrateddistances from the c entre of rotation. These holes serve as calibrations for measuring the distances in the analytical cell. At the beginn ing of sedimentation this peak of refraction will be at the meniscus. The Rayl eigh interference operates on the basis that the region of the solution in analytical cell harbour ing macromolecule will have a refractive index higher than the rest of the solution. as the macromolecules move down the cell the )eak also shifts giving direct informa tion about the To vacuum Counterpoise Lens Photographic plate Mirror Lens DriVe shaft Analytical cell Rotor chamber .Motor Centrifugatlon 307 refrigerated and has an evacuated chamber. With the progress of sedimentat ion. Lens Monochromator Slit Light source . The first three rotors are discussed below. have been measured by this techn ique. the tubes and the solution within also take the same angle (Figure 10. even when they are in a gross mixture. Analytical ultracentrlfugation has made it quite easy to measure the coefficients and molecular weights of macromolecules. measuring molecular weights. it is used quite frequently for analytical purposes too. Figure I(. This is so because apart from being used for preparative purposes. One point must be made clear here. and zonal rotors.3 (B) provides diagrammatic representation of analytical ultracentrifuge system. misnomer. swinging. Fted-ang/e rotors: These rotors have holes within their body and one can slide t he centrifuge tubes within these holes. Since the holes are at an angle (between 14° and 40°) to t he vertical.4). Roto Basically. Molecular weights species. it has several other applications discussed elsewhe re in chapter. The name 'preparative ultracentrifuge' is a b it of. rotors come in four varieties : fixed-angle rotors. bucket rotors.14° 40° 3o8 Bphysat Cherrs sedimentation characteristics of the macromolecules. vertical tube rot ors. The whole optical informati on continuously photographed. Under centrifugal Centrifugal field Tube angle Axis of rotation Centrifugal field . Zon al rotors will be l discussed in a later section. This will become clearer when we applications of this type of ultracentrifuge. [D) (E) Figure 10. (C) Ap plication of centrlfal fore leads to reorientation of the sample and the gradient. (E) Gradient reorients as the rotor stops. (A) CroSS section of a typical flxed-angle r otor. The flxed-angle rotor. (D) The sample components separate.4. . (B] If the centrifuge tube {s fllled with a gradient and placed in the rotor: no centrlfugal force ts applied. Howev er.filled with gradient and sample.5). see discussion under wall-effects).5. Sedimentation in these rotors occurs across the diameter of the tube. This sliding down makes the sedimentation quicker (wall-effect). (F) As the rot or decelerates.Centrlfugatlon 309 field. no centrifugal force applied. (For more. the particles move radially outwards. these holes lle parallel to the rotor shaft and n ot at an angle. (A) Centrifugal field J Axis ofrotation (B) (c) (El (F) Centrifugal field Figure 10. (D) The orientation that the gradient and the sample as sume under ccmtrlfugal force. the solution orients back to the original position (Figure 10. there is a severe disadvantage . The particles then slide down the wall and the pellet is formed at the out ermost point of the tube.particles differing highly in their sedimentation characte ristics can only be resolved in these rotors. (B) Tube. The partic les thus have to traverse the shortest possible distance and sedimentation is quicker tha n the other rotors. Moreover. As the rotor decelerates. Vertlcal-tube rotors: These rotors too have holes within their body in which one can slide the centrifuge tubes. travel only a short distance before they strike the wail. This makes rm quite la . As the rotor accelerates and centrifugal field is applied. The vertical tube rotor. (A) Cross-sectlon of a typical vertical tu be rotor. the tubes lie at the edge of the rotor. (C) Application of centrifugal jeld leads to reorientation of the gradient and the sample. the solution within the tube reorients through 90. the original orientation is r egained. particles which do not differ much in their sedimen tation behavior are not resolved. the gradient and sample begin to reorienL (G) When the rotor stops. However. This reorientation makes it lie perpendicular to the axis of rotatio n. (E) The sample components separate. When the rotor decelerates. This can be a disadvantage as the pellet may fall back into solution at the end of centrifug ation.rge and the RCF. Swfnglng-bucketrotors: As against fixed hole type rotors we have seen above. Even if it doesn't fall back. The solution in the tube reorients to lie perpendicular to the axis of rotation and parallel to the applied centrifugal field.6). This is anothe r reason why separation in these rotors is quicker. is more than what is possible in any other rotor. it may not be easy to reconstitute the whole pellet and some loss of yield may be inevitable. . at its minimum. The pellet in such.rotors will be deposited all along the outer wall of the tub e. the se rotors have buckets that swing out to a horizontal position when the rotor accelerates. the tubes fall backto their original position and the solution too regains its original orientation (Figure 10. however. (E) When the rotor stops.310 (c) (E) Centrifugal field Biophysical Chemistry Centrifugal field Tube at res Tube during centrffugation Axis of rotation F3ure 10. (C) As the centrifug al force is applied. Wall Effects or Trajectory of a Particle Inside the Rotor Tube The centrifugal force acts on the particle in an outward direction. the particle does not travel in a straigh t line towards the bottom of the tube. This results in a rap id sedimentation of particles in a fixed angle rotor tube. the bucket reorients to its original position. This is known as the wall effect. These rotors too suffer from wall-effects like the swinging bucket rotors. gives rise to strong convection currents due to which it is not possible to reol .6. Rather it moves outward within the tube till it hits its wal ls and then slides down this wall to be pelleted at the bottom (Figure 10. the particles strike the wails of the tube and s lide down. This behaviour. (B) Centre tube fllled with the gradlent and sample. Even in swinging bucket rotors.7). But t hese rotors are normally used for density-gradient centrffugation and the density gradient can c ushion and lessen the wall-effects. The swLnfling-bucket rotor. the tube swings out and orients at rjht angles to the axis of rotatlon. Thus. (D) The samp le components separate. when p erforming centrifugation in a fixed angle rotor. (A) Cross section of a ttjpical swinging -bucket rotor. no centrifugal force applied. ve particles which vary very little in their sedimentation characteristics.7 Schematic diagram of convectlon currents generated due to wall effec ts./ sedimenting (High eoneentration]/( particles id Thin layer of Pellet Aseending layer (Low concentration) Figure 10. The diagram shows parles descending along the wall of the tube (descending layer) . On the other hand. this rotor design is Axis of rotation Supernatant Cite: tee!e tube of fluid [I //1 Boundaxy of | Descending layer ---. This is because the particles in a centrifugal field. DIFF]INTIAL CENTRIFUGATION Recall equation (I0) from our previous discussion of basic principles of sedimen tation.. albeit to a lesser extent.. 9 rb t=--× In2 . Separations carried out in a suspending medium which is homogenous are known as differential centrifugation while those carried out in a suspending medium ha ving density gradients are known as dens/ty gradient centrifugations. fan out radially from the centre of rotation. rather than sedime nting in parallel lines. The discussion of zonal rotor is deferred to a later section as an unders tanding of density gradient centrifugation is essentiaJ to understand its working.slide down them to the bottom. Preparative centrifugation methods can be divided into two main techniques depending upon the medium of suspension in which the separat ion is carried out. However. This particle behaviou is true for swinging bucket rotor tubes also. the gradient usually decreases these convec tion currents due to wall effects. the best way to minimize the wall effects is to use a zonal rotor.Centrifugation 311 very helpful in separation of particles which vary in their sedimentation rates by a significant order of magnitude. PREPARATIVE CENTR/FUGATION Preparative centrifugation is concerned with the actual isolation of biological material for subsequent biochemical investigations. 1. in these cells also t he particles hit the wall and then . Thus. . Since swinging bucket rotors are g enerally used for density gradient centrifugation. Attempts have been made to' minimize wall effects by using a sector shaped cell in the swinging-bucket rotor. Similar data would also be generated for other i ntracellular organelles {all the data represented here is approximate and it varies with tiss ue and species differences}. upon subjecting the homogcnate to centrifugatlon for 15 minutes at 10.FIgure I0. This is the e ssence of differcntlal centrlfugation.000 g. the mitochondria and lysosomcs would pellet out. Similarly.The above equation makes it clear that a mixture of homogeneous partlclcs can be separated by centrifugation on the basis of their densities and/or their size. The best strategy that we can adapt now to separate the tissue hom ogenate into various organellcs is to centrifugally divide it into a number of fractions by i ncreasing the applied centrifugal field at each step. and the centrifugal field constant} we can arrive at a fair estimate about the t ime requlrcd for each of them to sediment completely in a given centrifugal field.8 provides an outllnc of a typical fractionatton proce dure based on differential centrifugation. This can be achieved either by the time required for their complete sedimentation in a fixed centrifugal field or on the extent of their sedimentation after a given time in a fixed centrifugal flcld.. If we assemble all the data so generated we would find that under a flcld of roughly 1 000 g the cell debris and the nuclei would sediment if the field-is applied for about 15 minute s. If we now substitute in the above equation the approxlmatc data about the shape and densities of various intraccllular particles {we keep the mcdinm and therefore i ts characteristics. The pellet and supernatant arc separated at the end of each step and the supernatant reccntrifuged to sediment another lighte" intracellular organelle. . In a similar m anner we can va the value of the centrifugal field applied for each intracellular organelle a nd again find out the time required for each of them to sediment completely under various centrifu gal fields. We can choose the centrifugal field in s uch a manner that a particular organcllc sediments during the already known time of centrifugation to give a pellet. l centratlon Every pellet has to be washed several times..Small sized particles .i.8 Outline of a typlcal fractwnatlon by differentW.e.des l. Small sized particles Large sized particles (i) Time of centrifuUon . it consists of contamination from the other pin..000 x g I00. This is so since the pellet obtaine d is never pure. apart from the desired particles. i. Large sized particles Medium sized particles .312 lO00'x g I0.000 x g Pt 5-15 rain 15 mln I-2 hr of microsomes Tissue Pellet of of (.'. Pellet of homogenate nuclei mitochondria + some heavy lysosomes solubles lgure 10.000 x g 100.. th yidof . Howwr.Resupenson ofthepellt and reeen yeld apellet which sfcdrly putw. and isopycnic centrifugation. convenience and time economy.contained in the homogenate. eliminate mixing of sepa rated due to convection and mechanical vibrations. For experiments which st rictly absolute purity of preparations an alternative method known as density gradient centrifugation (see below) is used. however. The separation centrifugal field is therefore dependent upon the buoyant densities of the parti cles. (i)). It pointed out here that the repeated washings invariably reduce the yield of the f inal Inspite of its reduced yield differential centrifugation remains probably the mo st commonly method for isolation of intracellular organelles from tissue homogenates because of its ease. gradient is reasonably shallow. where a homogenous medium is used for gradient centrifugation employs medium which has gradients. apart from exerting their separating effect. Centrifugation-res ults in sedimentation particles at their respective sedimentation rates till a pellet is formed at the bottom of the tube (Figure 10. 2. This method gives a much better than differential centrffugation. originally suspended near the bottom of the tube also and thus contaminated the pellet. The technique involves careful layering of a sam ple on top of a preformed liquid density gradient whose density continuously increas es .Centrifugation 313 .9.9 B and C) however. This pellet. DENSITY GRADIENT CENTRIFUGATION As opposed to differential centrifugation. 10. gradients. Centrifugation The gradient used here has maximum density below that of least dense sedimenting particle. To understand the situation let us take a look at Figure 10. yield a fairly pure pellet of large partic les. centrffugation. is not entirely made u p of the large some of the lighter particles.9A (ii) and (iii)). The drawback of this method is ofcourse its poor the fact that the preparations obtained are never pure. Before the centrifugation is initiated all the particles of the homogenate are h omogeneously distributed throughout the centrifuge tube (Fig. Density gradient centrifugation has two variat ions.9A. Repeated resuspensions and recentrifugations pellet (Figure 10. zonal centratton. Note that separati on s due to d{lerences in sze and shape. (A) Sample layered over a continuous densWj gradi ent.the bottom of the sample tube. The sample particles travel through the steep gradient a nd form zones depending upon their sedimenting rate. For separation to be achieved b thi s it is necessary that centrifugation be terminated before any of the zone reaches the centrifuge tube.. Centrifugation is then performed at a comparative ly speed for a short time.: Imnll portloloe Medium eled pmrtleloe . (B) Posgton of zones due to dterent sze of parcles at the termlnaton of centrtton.. This method is useful for separating particles which differ in size Sample containing particles of different sizes small particles medium psrtlolss largo pilrllolee A D .oLarOo pertleloe | O. . I 0 RaW. isopycnic method used gradients which are reasonably steep so that at maximum. will become zero. I summarizes the differences between the two techniques.314 Biophysical Chemistry but not in density. This is the essence of Lopycnlc centrg@atlon also known as the n eqW10rlum centr0at/o The method differs from rate-zonal centrifugation in two significant ways. Thus.10 illustrates the salient features of rate-zonal centrifugation. Table 10. different particles differing in their buoyant densities will travel different lengths and become stationary at a region where the density of the layer below them is greater than their own buoyant density. (il) As opposed to rate-zonal method where centrifuga tion is carried out for a limited period of time. If a densi ty gradient is now prepared in a tube in such a manner that the density goes on increasing towa rd the bottom of the tube and a solution of different particles is centrifuged in this medium. Isopy©nic Centrlfugation For better understanding of this technique. where the grad/ents were shallow. The method has been very usefu l for separation of RNA-DNA hybrids and ribosomal subunits. Figure 10. (i) A s opposed to rate-zonal centrifugation. while this method is extremely useful for separation o f proteins possessing nearly identical densities but differing only slightly in their molec ular weights.lcnic eenation Rate-Zonal Isopyeni¢ Synonym . Table 10.1 S--igniflcant differences between rate-zonal and iso. lysosomes an d peroxisomes which have different densities but similar sizes. it would be good if equation (9) is recaged In the above equation if one substitutes identical values for the density of the particle and density of the medium. . isopycnic method allows centrifugation for pro longed periods at relatively higher speeds to permit all species to seek their equilibrium densiti es. it is not at all useful for separation of organdies such as mitochondria. the gradient density is greater t han the most dense sedimenting species. the rate of sedimentation. high speed. ribosomal subDNA. plasmallpoproteins. sedimentation equilibrium. One way is to carry out a preliminary centrifugation of the sample at an RCF which will sedime nt particles heavier than the one desired. Gradient Shallow. Density equil/bration. The supernatant is then layered over a medium whic h has the same density (isopycnlc) as the desired fraction. discontinuous gradients. . peroxisomes etc. the desired fraction wiB-migrate to the middle of the liquid column. gradient sedimenting specie. There are two ways in which isopycnic centrifugation may be carried out. maximum gradient density less than that of the least dense greater than that of the most dense sedimenting specie. lysosomes. units etc. sedimentation veloc/ty. maximum gradient density Steep. short time. long time. continuous or continuous. mitochondria. while the one that is heavier will be at the bottom of the centrifuge tube. low Complete sedimentation till speed. Centrifugation Incomplete sedimentation.s-zonal. If centrifugation is now carri ed out. The fraction lighter th an the desired one will be at the meniscus. Separations RNA-DNA hybrids. equilibrium is acheived. very useful for separation of such intracellular organelles as mitochondria. and perox/somes which do not differ in size but differ in their buoyant densities. It is however. Table 10. Thus. No pretreatment of the sample is necessary and it can directly be layered on top of the densitygradient column. Upon centri fugation individual particles will sediment down the tube till they encounter a gradient whose densi ty is greater than their own buoyant densiW.2 Approximate densitie of particles in sucrose solutions Particles Denai (n/cm .II).2 lists the densities of a few important organelles and other macromole cules in sucrose soluUons. the method is not useful for separation of proteins many 0fwhich have similar buoyant densities inspire of differences in molecular weigh ts. The particles will thus become stationary forming distinct zones (Figure I0.rent zones n regions of the gradient havbg densities s bnilar to their Isopycnic separation depends solely on the buoyant densities of the particles to be separated and not on their shaPe or size. able 10. The gra dient should span the range of particle densities of interest. Srnlde cont|inlng clMere cleities Centrifugal force acting on the particle Higher density : acts as a cushion (A) The partles have formed d.315 The alternate way is to prepare a continuous density gradient in a tube. lys osomes. T he method is also useful for nucleic acid fractionation. 16 Intact oncogenic viruses I.I. 16 .30 1.60.70 . 18 Mitochondrla Lysosomes Peroxlsomes Plant viruses Soluble proteins Rhino.75 1.30.19 1.1.45 1.1.and enterov/ruses Nucleic acids.1.10 Plasma Membranes 1.Golgl apparatus 1.06 1.23 1. ribosomes Glycogen 1.30.16 Smooth endoplasmlc reticulum I.21 1.45 1. Apart from allowing the desired t ype of separation... (iO it should not interfere with the assay technique.3 Types of Gradient Materials Maximum density Sucrose (66%. (vii) it should be cheap and readily available. and (viii) it should preferably be recoverable for reuse . Sucrose is used routinely for rate-zonal centrifugation. Brakke (I 95 I) and Kahler and Lloyd { 195 i) were the first fe w scientists to use stabilizing gradients. A gradient material should meet several requirements. (vO it should be easily sterilizable. (iv) it should not absorb in the ultraviolet range. . There is no ideal all-purp ose gradient material. Sucrose still remains the materialin most general use. the gradient material should have the following properties : (0 it should not affect the biological activity of the sample being Separated. Table 10. (v) it should be non-corrosive to the rotor.5°C} Silica sols Glycerol CsCI Cs acetate . Table 10.. (tit) it should be easily removable from the purified product. while cesium chloride may be the usual choice for isopycnic centrifugation.3 provides data about most commonly used gradient materials along with their maximum densities at 20°C. They used sucrose for these gradients.Materials 316 Biophysical Cherntstnd Gradient Materials Pickels (I 943). 33 1.1 1118 NA NA NA NA 1.49 1. .57 1.63 1.91 2.Cs formate Flcol Sorbitol Renografln Urograffin Polyvinylpyrrolidone Diodon RbBr RbCI K formate Na formate Metrizamlde 1.37 1.37 1.45 NA = Not Available.0 2.26 1.32 1. sucrose interacts with the particles being s eparated and can ecert pronounced effects on the results obtained.Among the routinely used materials. The interaction varies from one type . Metrlzamlde has a disadvan tage in that it moderately quenches scintillation cocktail necessitating some correction when se parated components are to be assayed by scintillation spectrometry. They are also useful for separation of lipoproteins. Discontinuous gradients are especially useful for the of whole cells or sub-cellular organelles from tissue homogenates. or ¢ntinuous density gradient. modifying the buoy ant density of one with respect to the other. In addition many commercial preparations of sucro se contain ribonuclease and absorb UV light. Other popular materials are Picoll (high molecular weight sucrose polymer and eplchlorhydrln). an : incorrect choice of gradient material can lead to a situation where inherently large differences between the densities of two particles can be lessened. discontinuous or 'step' density gradient . Different particles react differently with different gr adient materials and consequently possess widely varying densities in different gradient material s. leading to a less than s atisfactory of Density Gradients Two types of gradients may be prepared. A correct choice of the gradient material would therefore be able to accentuate even small differences in densities between two particles leading to their complete purification. These impurities have to be removed before the gradient is prepared. The sample is then layered at the top layer and the tube is spun under the experimental conditions. Ludox (silica sols) and Metrlzamlde. Selection of correct gradient material is very important for a satisfactory sepa ration by gradient centrlfugatlon. Non-membraned solid particles are not affected as much as the membraned particles containing a large percentage of water. Discontinuous density gradient (where the density increases abruptly from one la yer to may be prepared very easily by carefully layering solutions of continuously decr easing over each other with the highest density solution being placed at the bottom of the tube.Centrfugatlon 317 of the particle to the other. This fact might be u sed successfully separate particles of overlapping buoyant densities by deliberately using a medi um which interacts more with one kind of particle than with the other. Continuous gradients (where the density decreases linearly from the bottom of th . On the o ther hand. the dense solution drips into the centrifuge tube. The level of the first chamber thus decreases and is compensated by the flow of the less dens e the second chamber. stirs solution of two different densities and the chamber now contains solution of a d ensity 'less than it originally had. The discontinuous layers slowly merge.e centrifuge to the meniscus) may simply be prepared by allowing the discontinuous gradients to stand long time. to give ris e to a lindar gradient. However. This chamber is connected to a second chamber s less dense solution. ribosomes . The first chamber has some stirring device. When the tap of first chamber is opened. this method takes very long time if viscous solutions are use d in of gradients since diffusivity decreases with increasing viscosity. gradients are useful in the separation of many proteins and enzymes. Altematively use of a special device may be made of to prepare density gradients of a nature (Figure 10. In this way a continuous linear gradient may be formed. The stirring device. The chamber which drips into tube contains very dense solution. The device has two chambers. through diffusion.12}. Thus the solution which drips into the centrifuge tube is of decreasing density. which has already been activated. The other chamber has solution which is less dense.12. The device has two interconnected cham bers. Thus centrifuge tube s of inner diameter of 2. a broad applied through a syringe which is held about 2-3 mm above at an angle of 45° touching the wall of the centrifuge tube.6.5 cm of sample. Recovery of Samples . The wall of the centrifuge tube without disturbing the gradient. L esser volumes of sample will not affect resolution but might be difficult to detect after sepa ration. The sample may be gradient inclined sample trickles down the like DNA.Dic@ram of a gradient maker. Sample Application to the Gradient The volume of the sample that can be applied to the centrifuge tube depends upon the cross sectional area of the gradient exposed to the sample. mouthed pipette is used in place of a syringe. will result in loss of proper resolution through broadening of the separated zones.318 Stirrer Biophysical Chemistnj Dense solution of gradient material Less dense solution of gradient material Density gradient Figure lO. Large volume of sample. The chamber through which the solution drips in the tube contains very dense gradient material solut ion.5 cm will accommodate only about upto 1 cm3 while tubes with diame ter of 1. em will accommodate only about 0. The general rul e is to apply a sample concentration which is about 10 times less than the concentration of the starting gradient. The concentration of sample that can be applied is also limited. if adde d. If the cent rifugation has been carried out to obtain sub-cellular organelles. The second method is known as the displacement technique. dis places the gradient layers which start coming out from the outlet provided in the cover (Fi gure 10. Through another hole in the cover a tube is p assed to th bottom of the tube. the presence of these ca n be assayed by measuring the activities of marker enzyme specific for each organelle. . A cover with an outlet is placed at the top of the gradient. A peristaltic pump might be employed to suck off the medium from the hole at the bottom.Two methods are in vogue.13B) and can be collected in separate tubes.13A). when it reaches the bottom.solution (more dense than the highest density i n the is pumped through this tube. The fractions so collected can be analysed for separated components. The first involves puncturing of the centrifugation tu be at the bottom with the help of a needle and collect the dripping medium in separate tub es in fractions of roughly equal volume (Figure 10. A very dense. The dense solution. can be shortened by using half-filled tubes. The three layers of gradient have been assigned arbit rary density values. Since rate zonal separations are not based on the densities there is a danger that such reorientations may disturb the separated It is true that longer pathlength of the cell means a longer time for separation . In this case not onl y is the .Intermediate density (30%) U 1 4----. However. (A) The bottom of a celluloid centrifuge tube i s pierced with a hypodermic needle. swinging bucket rotors give better The reason for their being suited better to this technique is the longer pathlen gth that rotor offers. The fractions dr/p into a series of tubes.Sample zone Highest density (45%) Solution density (60%) (A) (B) I O. the wall effects are considerably less and the contents do n ot reorient acceleration and deceleration. Also. 13 Sample recovery. (B) Collection of samples separated on a discontinuous gradient by displacement technique. A higher density solution when ptunlaed into the tube displaces the gradient whi ch is collected into of Roto If the method adopted is rate zonal centrifugatlon. time required . Fraction density (60%) [ c°llect°r1 Least density { 15%) Sample zone 4----.3i9 Solution [-. If.4 and 10. since the is on the basis of density and each component becomes immobile at regions equal to the reorientation of contents at deceleration is not a great disturbing factor. in Zonal Rotors In a previous section we have already discussed that the centrifugal force acts in an making the particles move radially in both the fixed angle type or the swinginggives rise to strong convection currents due to wall effects. Both these contribute to limiting the time and as such rate zonal separations can be achiev ed in required for a fully filled cell. however. The reason for this is tha t the in these rotors is banded over a large surface cross-sectional area. the tube rotors have the shortest pathlength and as such sedimentation is quickest i n rotors. fixed angle and vertica l tube bring samples to equilibrium in a shorter time as compared to the swinging bucke t It should be clear from Figures 10.5 that as the rotor tube angle decre ases. The particles thus have less distance to travel. . Fixed angle and vertical tube rotors separate sample zones better than swinging bucket the technique being used is Isopycnic centrffugation. Naturall y. As would be clear from the earlier discussion on rotors.shortened but the centrifugal force at the meniscus also increases. Also. the length also decreases. 14A).320 Biophysical Chemistry centrifugation is carried out in a sector shaped cell the particles will not mak e a contact the wall even when they move radially (Figure 10. Figure I0. is not the main reason behind centrifugation. G-Channels through which gradient . This is made possible by what is known as a zonal rotor. however. 14 (A) Sector shaped cell of a zonal rotor. the zonal is more or less a flattened sphere which has its interior subdivided into four e qual (sector shaped) by means of a four. Initial position of sample Positions of separated components Figure I O.veined core. As evident from the figure. and (iO sample port. limitation can be understood more clearly if we consider the follo wing example. Arrows indicate ports at the base ends of the veins. This pr oblem be solved by using zonal rotor which was first developed by Anderson and his col leagues.14B shows the design of a zonal rotor. However. (B) Design c (B-XV). Wall effects do not pose a problem. The main reason behind the use of such rotors is the small amoun t of that can'be accommodated in conventional rotors during density gradient the largest swinging bucket rotors have a combined bucket capacity of only I00 m l.15 Cross sectlon of one quadrant of a zonal rotor showing {i) the special holes drilled into the sepia for gradJent loading. it very difficult to prepare sufficient quantities of materials required in many experiments. Figure 10. Note the four veined core. Minir01zing convection currents. the amount of glyoxysomes req is tremendous and it would require many days to achieve the quantity by using swinging-bucket rotors and the density gradient methods described above. To purify enzyme citrate synthetase it is necessary to make a preparation of glyoxysomes a rich source of the enzyme in question). e. i.15). The gradient can thus be pumped into this special seal and bearing assembly from where it will reach the sector shaped quadrants while the rotor is moving (Figure I0.opposed to conventional methods. without a density gradient. When the speed has been brought up to 3000-4000 rpm.. the gradient is loaded the rotor (dynamic method). This is possible because the four veinings of the core are hollow because of the holes drilled into them. Again unlike conventional methods (in which the most dense solution is poured first) the gradient: in a zonal rotor is established by pumping the material of the lowest density first. In the . the zonal is rotated while it is still empty. These holes are continuous with the holes onto the perlmete of the rotor's interior and with the special movable seal and bearing assembly. The last to be added is the densest materia l own as the fluid cushion. the equations considered so far do not take into account th design of the centrifuge. let us turn our attention to the analytical applications.ary during sedimentation. the movable seal and bearing a ssembly is dismantled andthe rotor chamber is closed and evacuated. all the equations we have considered so far are for spherical and unhy . There are quite a few factors affecting sedimentation that we are yet to address . Utilizing these holes (sample ports) the sample material can be loaded on to the top of the gradient so that it is very c lose to the centre of rotation. the concentration of the suspension. Let's first deal with 'boundary sedimentation. All these com plex factors do affect the sedimentatlonproperties of a given particle. This operation contues l the entire sector shaped cavity is. Secondly. It is called boundary sedimentation because in this form of centrtfugatton the initial position is a homogeneous sol ution. and the nature of the medium.A boundary is thus formed between clear solvent and the region where the particles are pres ent. As the ¢entrifugation starts. First and foremost.) Having seen the preparative uses of centrtfugation. Much information about the particle is now obtained by following the progress of this bound.Centr/fugat/on 321 subsequent sgcs materials of increasing density arc pumped which displacc the lo west density layer toward the centre of rotation.' a name wh/ch is a variant for differential centrlfugation that we have dealt with above. This operatio n displaces the gradient along with the separated sample components out through the central part s previously used for sample loading: The effluent can be collected as equal fractions in a s eries of tubes. When the centrifugation is over a yery de nse solution is pumped through the veins to the periphery of the rotor's interior. BASIC PRINCIPLF. Subsequently the rotor speed is increased to the desired level where the sedimenting species will move radially till they reach a density which is similar to their own. Once the sample loading is complete. There are four more holes present at the base of each of the four veins. fu. ANALYTICAL CENTRIFUGATION 1. OF SEDIMENTATION (CONTD. particles start to sediment leaving a clear layer of solven t above. it berg we asec. is ratio is clor to 1( I to 1. To arrive at a mathematical expres sion for the sedimentation rates of such particles. fifo is e con rao for a ven molle. that we have been considering in the above equations. e rao be nsiderably g. equation (9) is suitably modified to give dr 2 r {f wherefis e cflo coecient for e asec or hydrated pcles dfo is e fcon coeent for e unhydrated. ge ts replaced by the effective radius or the Stokes radius. the actual radius of the particle. e ratio be lge. sphec pcle. . tg w dnt der s. for eple e obul proteus. What if the particles are not spherical and/or hydrated? In such case s. For pcles wch e more or less sphec. r-e nature. e c e of s t may not become eately cle. For fibrous proteus. But let's consider a protein its native stat e d e e prote e denated ste. og to eff long. For DNA too. e confoaons ese o states der dely. The asymmetric particles sediment at a lower rate owing to the larger friction they experience.4). e above reflonsp esmbshes a we pot point : ff s s s b ss et.drated particles. ct of Concentration: Consider macromolecules which are very large and asymmetr ic (extended}. Let us consider a better example. An enzyme is supposed to change its conformation subtly when a true substr ate approaches it. Thus. and a more usefu l one at that. H owever. an enzyme without substrate. This relationship then becomes the principle on the basis of which one can explo it centrifugation for studying the conformation. It is also important to consider that the solvent molecules are much smaller com pared to the particle. In the above discussion it has been mentione d that the shape of the molecule largely decides the sedimentation rate of that particle.322 Biophysical Chemistnj The above example is extreme. Imagine that they are rotating. it is largely affected by the solvent( see chapter 1. Another factor is the charge t hat the molecule may carry. and an enzyme with the substrate may have sllghtly different conformations. Now as these macromolecules rotate. These factors need to be discussed. and. This rest riction on the movement of solvent molecules around the approaching macromolecules means that t he effective . and between the particles and the environment --that affect the sedimentation behavlour of t he particles. As concentration of such molecules increases. changes in the confor mation of a given macr0molecule. These charges may interact with each other and modify the sedimentati on behaviour. Yactors Affecting Sedimentation Velocity We have already discussed the particle related parameters and the environmental factors that affect the rate of sedimentation of a given particle. they will occupy a very large volume of solution. It is very difficult for the solvent molecules to move in the opposite direction. However. t he frequency of collision also increases. this shape will be further deformed. the shape of a given molecule is not a static characteristic. They will then sediment at different rates.at high concentration. indeed. Also ignored so far is the fact that when a molecule is made to move under a brute force. Moreover. and 8). F. Further imagine that these molecules approach each oth er. the particles may themselves i mpede their own migration! A brief discussion of these factors is given below. there are ot her factors factors which arise due to complex interaction between the particles themselves. the concentration of the part icle may itself affect the sedimentation . with everything else constant. and the ef fect of sedimentation velocity also will be substantial. However. Please note that in equations (8). and (I0) th e term for viscosity does not include this modified viscosity around the macromolecules. DNA is a very extended molecule. In su ch a mixture. the chances of collision will be that much more. The sedimentation velocity will decrease definitely but to a small e xtent. the viscosit y around it will be largely affected. Now consider two examples. concentration dependence of sedimentation velocity increases with the size and asymmetry of the macromolecule. In the second case though. They will occupy less solution volume since they are not extended. everything impedes the sedimentation of everything else. Consider a situation where you are sed lmentlng a mixture of macromolecules. some very large and some small by comparison. Thus. in the first case the molecules are largely spherical. Thus. the things will be entirely different. The larger molecules im pede themselves but they also have to move through a solution of slower-moving molecules. (9). as the concentration of this molecule is increased. Then there is the Jhonston-Ogston effect.viscosity is higher around the macromolecules. The effect on viscosity of the medium around them will be quite less. At hig h concentration . Naturally the sedimentation rate of the macromolecules will be less. (I) What happens to the sedimentation velocity of a g lobular protein when its concentration is increased? (2) What happens to the sedimentati on velocity of a large DNA molecule when its concentration is increased? In both the above cases the chances of collision will increase as the concentrat ion rises. This effect becomes more pronounced as the molecular size increases. This can lead to aggregation and fo rmation of a macromolecular cluster that will sediment at a higher rate and pellet quickly. This problem can be done away with by carrying out sedimentation at higher ionic strength . such aggregation can occur at high speeds even if one is working with very low concentrations. Thus. A small time after starting sedimentation. F.negative charges in front and positive way behind. However. In extreme cases. The neutralizing counterions of the solution are very small as compa red to the macromolecules. Fect of Speed: From the equations we discussed above it would seem that the rate of sedimentation should be proportional to the centrifugal field. For very large macromolecules. And even then the experimental results may not be entirely factual. a peculiar situation may result. If sedimentation of such macromolecules is carried out in solution of low ionic strength (0. a clear separation of result . this effect is extremely high. the macromolecule moving ahead leaves a wake behind it. it is very necessary that sedimentation be done at very low concentrations. the entire mixture may move at the same sedimentati on velocity and hide the fact that there are more than one species having different sediment ation rates. If the spe. In view of such anomalies.Centrlfugation 323 then the larger molecules are so much impeded that they might move along with th e smaller molecules. It is therefore absolutely essential that one works at low speeds if correct results are to be obtained. It is obvious that the ionized macromolecules will sediment much more quickly than the small counterions. A potential gradient would thus get created such that it is against the direction of sedimentation. for chromo somes.01 M).ed is quite high. Consider that the macromolecules bear a negative cha rge and the counterlons are all positive. this may not be so always. T his effect can slow down the velocity of the macromolecules.ffe¢t of Charge: Biological macromolecules are usually charged. Let us see what may happen here. At high concentrations such proportionality may not exist for macromolec ules. This is especially true of DNA owing to it s large size. This wake can accelerate the macromoiecule moving behind it so much that it can reach the macromolecule moving ahead. the sedimentation rate will become proportional to c02r. and the density. sedimentation rate studies are performed using a wide varie ty of systems. It is not surprising then that velocityexperiments are carried out with a purpose of determining s.(13) Look at equation (13).. it will be obvious that if the env ironmental are kept constant. In the above discussion we have taken the composition of the suspending medium t o be However. It should be fair ly apparent In view of the environmental factors being kept constant. s Is a measure solely of the particle rethe size. SEDIIENTATION COEFFICIENT The velocity of a particle per unit centrifugal field is referred to as its sedi mentation It is denoted by the symbol 's'. The mathematical expression of s is therefore V s = . the shape.. in reality. If s is to reflect properties due solely to the particle.. then we must find way to correct the experimentally determined values to a constant environmental set. v = s t02 r . Compare it with equations (9) and (11).2..(12) 2 0r Now if you reconsider equations (9) and (11). The . o the viscosity of water at 20C. Factors Affecting Standard Sedimentation Coefficient Mo/ecular Shape: Lrge. the/r entation coefftcient /ncrea e markedly as the friction decreases. For convenience the value of $ is usually reported in svedbergs (S) and 1 svedberg is taken to b e equal to 10-Is seconds. exended molecu/es wil/ce more cflon wh/le the sphe rlcd molecules face less riction. .w is dubbed as the standard sedimentation coec/enL T is the viscosity of water at temperatur e T.¢14) where so is the experimentally obtained value of sedimentation coefficient and s o. This is done by subs tituting the observed value of s into the following formula 2o o (1-v) . By convention.. solution vi scosity and density. The bases in double stranded DNA exist in a highly stacked manner. rigid.62 ' 324 Bbphyslcal Chemistry The experimentally determined value of s is affected by temperature. These two factors are primarily responsible for the rigidity of DNA. c the viscosity of the solvent at a given temperature and o the viscosity of water at that temperature. S 2o.. However. and is the partial specific volume of the particle. the bases of the two strands are in contact with each other through hydrogen bonds.w is sedimentation coefficient that would be obtained with water as solvent at 20C. Thus when we say that the prokaryotic ribosome is 70S. what we mean is that it has a sedimentation coefficient of 70 x I0-s seconds. The higher ction in rod-like molecules is because t hey cannot bend.w is the density of water at 20C. the observed value of s is corrected for that value whic h would be obtained if water was used as the suspending medium at 20° C. It is normal to keep the temperature of the solvent as close as possible to 20C. This is best illustrated in the example given below o f DNA denaturation. if the/r rigidity is destroyed. P. PT is the density of the solvent at temperature T. Moreover. Most values ors for biological particles lle between 10-I" and i0-n seconds. 16 A plot of sedimentation coe. Since . Heating tends to break hydroge n bonds.40 44 48 52 56 60 Temperature (°C) Figure 10. Note that the value of s has incr eased b{1220 at the h@hest pot. DNA was heated in a solutlon of pH 7. the solution was cooled to 20°C for centrlfugatlor Formaldehyde was added to prevent r eformation of hydrogen bonds when the solutWn was so cooled.S and O.01M wtth respect to PO c After heatlng. Imagine a long DNA molecule that is being heated.ent values of T7 DNA of molecular weigh t 25 million as a functlon of temperature. In the regions where the hydrogen bonds are broken. the bases become less stacke d. .16 describes only half the experiment discussed abov e. the sedimentation coeffcient of double stranded DNA should increase.(15) This relationship is valid for double stranded NaDNA at neutral pH in 1 M NaCI w . it should feel less friction and its sedimentation coefficient should increase. After several studies the relationship between s and M for DNA was found to be s2o. This is done because it is simpler to determine the former .3. the DNA mol ecule can bend at these points. This is exactly what is observed in an experiment like this (Figure 10. Molecular weight: Figure 10. This happen s because the molecular weight is suddenly halved (Figure 10.16). 40. Naturally.17). As the temperature i ncreases.8 + 0. The moment this happens..w = 2. Thus with an increase in temperature.17 Continuation of the experiment described by the plot in Flgur 10. Now. Also evident from the above two plots should be the fact that frictional coeffci ent and molecular weight are very related functions. becomes a study to find out the relationship between frictiona l coefficient and molecular weight.Centrlfugatlon 325 both the rigidity imparting factors are undermined in these regions.44148 52 56 60 Temperature (°C) Figure 10. all the hyd rogen bonds between the two DNA strands are broken and the molecule is rendered single stran ded. The sedimentation coefficient is then supposed to reflect the molecular weight of the macromolecul e. Previously the molecule was fully extended. owing to loss of rigidity at places. This is enough to indica te that s is dependent upon molecular weight of the molecule. The dashed llne represents sepcwatlon of strands. If the temperature of the solution is allowed to increase continuously. in fact. Such studies become especially important since many of the res earchers prefer to characterize a macromolecule by Just finding out its sedimentation coe ffffcient and not its molecular weight.00834 M°'497 . more regions where the DNA can bend are created and the molecule becomes progressivel y more flexible. the molecule can bend and its extendedness is reduced. Thus any study to find out the rela tionship between s and M. drops precipitously. the curve that was rising. For proteins. the situation is qttite different to that of DNA. Thus a slight error in calculating sedimentation coefficient is reflected in a molecular which is Just slightly off. Here s varies as M/. .hen the sedimentation is carried out in a sector shaped cell and if the s value lles bet ween 10 and 60 S. This is so since a small error in the measurem ent of sedimentation coefficient can be amplified and a value of molecular weight so ob tained will be oil significantly. Although the relationship exists. however. it is not very good to calculate the molecular weight from sedimentation coefficient values. For example.. Much more significant is the change in the shape of macromolecules that can resu lt asa consequence of solute binding. Double-stl'd ÷ .001 0.(12) 30. 20.001 M NaCl). if the ionic strength is high (0. On the other hand. Here. the negative cha rges due to phosphate may be neutralized by the counterions in the solution and the DNA may collapse into a random coll. such a change is a function of the flexibil ity of the macromolecule.. The se negative charges repel each other and keep the molecule extended. 0. it has a higher molecular weight and the refore a higher s value.001 M and a 0.05 M solution of NaCl. In the latter. In a solution of low io nic strength (0. DNA bears negative charges due to the phosphate groups which are ionized. s values for a single stranded DNA are significantly different f rom each other when the ionic strength of the solution differs. The double stranded DNA molecule is not only very extended but is very r igid also. On the other hand. The random coil is more compact as compared to the extended DNA structure and therefore sediments at a rate faster than the latter (Figure 10.18). Thus there is a difference in s valu of the same DNA if it is suspende d in NaCI and if it is suspended in CsCl. there is virtually no difference bet ween the s values. if sedimentation coefficient of double stranded DNA is determined in a 0.. The molecule thus carries a large negative charge.05 M NaCI).01 326 Biophysical Chemistry Sol.t Bind|rig: Binding of small solute molecules to large macromolecules affect s their s values. these negative charges cannot be overcome and the molecule remai ns extended. 0.19).. In addi tion we have also seen other conditions which affect rate of sedimentation. which is. Doubl e stranded DNA.flexible.flexibility of the macromolecule. Measurement of Sedimentation Coefficient Recall equation (12) v We have seen earlier that v = dr/dt.. These condit ions also affect . Since to2 is decided by the investigator. is almost unaffected by an increase in NaCl concentration. On the other hand the s value of single stranded DNA. which is rigid. We have seen above the conditions that affect sedimentation coefficient.I NaCI (M) lgure 10.18 Solute effects depend on the. the slope provides a determination of s.05 0.(16) t0r 0 dt A plot of log r versus t produces a straight line whose slope will be t02s (Figu re 10. undergoes a large change with increment in NaCl concentration. Therefore dr/dt I d In r 2 2 . clear solvent is left behind at the meniscus. Initially. mea surements are done at afleast four different concentrations. ffabsorptlon or refraction of light is measured at this point of time. the particles are distributed uniformly throughout the solution. Thus.(C} Centrifugation 327 the sedimentation coefficient. Concentration affects sedimentation coefficient. the upper flat portion is called the plateam (C) Concentration gradient.* Time (minutes} lqgure 10. the param eter now differs at places where there is clear solvent and where the particles have migrated. Concentration Distribution Each component sediments at a rate dependent on its sedimentation coefficient. (B} A plot of concentration vs.19 Plot of log r vs. The pH must be controlle d within suitable limits with the help of a buffer.20 Concentration distribution after a given period in a cell fllled with a homogeneous solutn. Thus to avoid charge relat ed effects. we have to take care to minimize the effect of these factors. this is what is obtained with scldieren optics. In the initial Fure 10. mea surements are performed at several speeds. time in minutes. We have seen that the best results can only be obtained at very low concentrations. As the sedimentation starts. distance: the region where the oonc entration changes is called bounda. Once this happens. (A) Poslt2on in the cell.05 to 1. the ionic strength of the solution must be between 0. When we measure sedimentation coefficient. the whole cell (the di scussion here pertains to sector-shaped cells since wall effects are almost non-existent in th ese ceils) should give the same data. in boundary sedimentation. To nullify speed related effects. Ther efore. T hus. we should see the following situations as the centrifuga tion progresses. Wh ether one is measuring the absorption due to the particles or the refractive index. plot of derfvata of concentratgon.0 M. . theref ore. The slope is used to arrive at t he value for s. the particles start moving towa rds the bottom of the cell. . the sedimen tation coefficient will differ.zero concentration. Thus. at. however. is o is measured at several concentrations and extrapolated to an experimental impossibfllty. ff any. )" This. Here s knfln/t¢ dflutlons to obta/n a value of so.. Moreover.* True sedimentation coefficient can only be obtained at zero concentration (s 2 0. one should also remember that the sedimentation coefficient that has b een deterrnined is of a macromolecule that is bound to counterions and to ligands. de pending on which counterion has been used and how much it binds to the macromolecule. There are two features t o this plot: the distance sedimented and the height of the boundary. Figure 10.sed throughout the cell. Figure 10. Now the cle ar solvent refracts or absorbs differently as compared to the region where the particles are present .materials. But here there are two components n the so/utWn. The boundary that is more to the bottom has a ne that is to the left has a lower s.20 is for the simplest situation possible : all the particles in the c ell have a Single sedimentation coefficient. 20 ). Thus. Since there . This p lot is what is obtalnedwlth schlieren optics or other monitoring techniques. all the particles are of the same kin d. this bounda ry will move toward the bottom (Figure I0.21) are being sedimented. M 328 Biophysical Chemistry conditions when the particles were uniformly dispel. The region where the concentration changes is the boundary. The s values of both the components are quite dljferent from one another. there is a boundary now. The distance sedimented is a measure of s and the height gives the concentration of the material concerned.20 (B} is a plot of concentration of the material being sedimented versus distance travelled.21 Same asia. Let us consider a cell in which two materials with distinctly ation coefficients (Figure 10. Figure 10. no boundary could be seen because the entire solution refracted or absorbed similarly. at the interface. What if the system contains a mixture of many components which have sedimentatio n coefficients differing only slightly from each other? We do see a boundary which is diffuse . or the concentration gradient. The area under the peak in this plot gives the concentration of the component. 10-20. The heights of the boundaries tell us ation of the two different sediment are two components higher s and the o about the concentr {A) {B) Figure 10.20 (C) is a plot of the derivative of the concentration curve. we obtain two boundaries. As the sedimentation progresses. in other words. 22 Concentran dtrbutlon m a cell contoJnm a heterogeneous solution of se veral component slhtly n their s values from one another.(Figure 10. lure 10. .22). the sha rper will be the peak. What information do you see in these figures? Don't read the in the text given below.23 Concentration distribution of different kinds of solutions after a given .10.the result will be a sloping curve (4) although we have not mentioned it so far. F.329 Distance Distance Distance Centrifugation From the above discussion following points emerge: (ll (2) a homogeneous solution gives a very sharp boundary. With these points at the back of our mind let's do a bit of exercise and then se e the applications of boundaIT sedimentation. the sharpness of the peak is also a function of the diffusion coefficient of the component -. Then check with the explanation. First make a mental assessment. A lso. Figure I0.the less the diffusion. the . the components have sedimentation coefficients quite distinct from each other. two sharp boundaries will be produced wh ich will look like steps. (3) if the solution has mmly components differing little in their sedimentation coefficients.xplanation in the text. the boundary will have many steps fusing into each other -.23.period of time. Look at Figure .23(A) tells us that there are four components in the material being se dimented. 10. if the solution contains two materials. component has a concentration higher than the other three. it is different from I0. Make a plot of the derivative of the conce ntration for this too. From this curve how can you tell whether it is a large diffusion c oefficient has broadened the peak or it is due to the heterogeneity of the mixture? Figure I0. It also is a heterogeneous mixture of many components. Figure 10.23(B) in the fact that there is one component in this se t which a sedimentation coefficient far removed from the rest of the components. In what would it be different from the others? .23(C) is quite interesting. This Co mponent faster and the rest of the components differ little in their sedimentation coeff icients and behind. The component moving ahead has a sedimentation coefficient higher than t he rest Make a plot of the derivative of the concentration curve for this too. The other three have similar What is not provided in the text and what you have to do yourself is to make a derivative of the concentration curve.23(B} is a heterogeneous mixture of many components whose sedimentatio n differ only a little from each other. This tells us that nucleases require divalent cations for thei r action. These components are actually the'broken pieces of D NA created by the nuclease action. Thus. of the mass of the total material. Figure 10. other studies we confine. is called a and it is a fraction f. the one corresponding to the intact DNA. that it is actually the magnesium divalent cation that they their action.24 Effect of EDTA on endonuclease activity. As nuclease activit y is started. The faster moving component is the intact DNA. All DNA has the same . There are now two components very clearly visible. the whole scenario changes. the endonuclease treatment has not begun. 3. the plot changes. we continue to observe essentially a single peak. the heights of the tell us the ratio of the Concentrations of the components. if in Figure 10. APPLICATIONS OF BOUNDARY SEDIMENTATION Proof That M÷ is ReqIred for Endonuclease Activity If double stranded DNA is incubated with an endonuclease. strand breakage may oc cur. As more time is allowed for nuclease action. all DNA gives essentially a single that all DNA present has the same sedlmentatlon coefficient.24. With this in the background let us see some applications of boundary sedimentati on. such a DNA solution is sedimented. (A) No nuclease added. If at zero time we put EDTA in the DNA solution and then begin nuclease action. No matter how much time we allow nuclease to act. EDTA is a go od chelator. And since concentrations are additive. one component i s moving and the other component is actually a mixture of several species havingve ry close sedimentation coefficients. Here b is the faster moving component.23(C). the concentration of the faster moving component decreases as we can see in the figure by the diminishing height of the boundary due to it. the data we get can be summarized in Figure 1 0.330 Biophysical Chemistry We have already seen that the heights of the boundaries tell us of the concentra tion of the components. then f = /(a + b). divalent cations. and Jacob wrote a truly prophetic paper proposing what we call today the general theory of allosteric regulation of enzyme action. that the active site is quite different from the regulatory site.ooe. Slow moving component seen which must be a mlxtt we of many different sized fragments. Only a sL'ue species of DNA ts present. Direct confirmation of the model came from the stu dies by Gerhart . EDTA inhibits nuclease action. At the time they proposed the theory. and that the sites for the regulators may be situat ed at different places in the enzyme. that there could be two differ ent kinds of regulators .inhibitors and activators. Proof for General Theory of Allosterism In 1963 Monod. (D) Nuclease added after EDTA has been added at zero tine. (C) Incubation for a longer time with endonuclease re sults in further: fragmentation of DNA. Changeux.ienL (B) Endonwlease added. they suggested. Making studies on the enzyme threonine deaminase their model. among other points. the evidence support ing them was fragmentary and inconclusive. Most of the ev idence was obtained from sedimentation studies on this enzyme. This is clearcut evide nce that the ee consisted of at least two different subunlts. only one boundary was seen. the boundal3. other boundaries appeared. When the enzyme was not treated with PMB.25 were obtaine d.6 S and the other at 2. as the con centration of PMB was increased.onthe0faspartatetmnsautj/aseTCase)atostdmn/ts. due to the intact enzyme completely disappeared and in lleu of that tw o boundaries appeared.2. the enmjme . The molecular weight studies (again with the help of sedimentation) provided that the larger s ubunit had a molecular weight of 96000 and the smaller one of 30000. one larger than the other.0 2 x 10-4M 4x 5. Sedb'n entolWn is from the meniscus on the left to the bottom of the tube on the right.25 ZJectofp. One sedimented at 5. However. At PMB concentration of 14 x 10 -4 M. PMB PMB 10.6 S B x 10"4M x 10-4M 14x 10-4M Centrifugatlon 331 and Schachman on the enzyme aspartate transearbamoylase (ATCase).8 S. The above sbc flgures pertain to ultn:wentrfge sedlJrwntan velocity runs. When Gerhart and Schachman treated the enzyme with p-mercuribenzoate (PMB) and subjected It'to sedimentation. the results depicted in Figure 10. When no PMB is pres ent.8 S 0. rne splits nto two peaks wn sedlmentation oefficlents of 2. As the concentration of PMB creases.sediments as a single peak. gradually the e nzj. Enzyme studtes with the isolated suburdts provided the following data. The smaller suburt . At 14 × I 0 M.6 S. The large r suburt stJll bnd to the substrate and carry out catalytJc activity. it could n o longer CTP like the tntact enzyme did (CTP is an trblbttor of the ev. the enzyme seems to have fu dia to ts subuntts. However.zyme).8 and 5. ivity. He proposed th at both the substrate and the enzyme changed their conformation as they approached each othe r. This was total confirmation of the ideas that Jacob and Monod had mooted.t. For decades scientis ts had believed in tlie Fischer's lock and key theory to explain the phenomenon of enzyme-substr ate specificity.26 depicts the effect of succinate concentration onthe sedimentation c oefficient of ATCase.03. The figure makes i t distinctly clear that as succinate concentration rises beyond a critical point there is a s teep change in the sedimentation coefficient of ATCase. This then is the direct prod for a conformational change in the enzyme upon addition of the substrate analogu e. importance in biochemistry : Koshland's induced-itt theory.the correct substrate could fit the enzyme active site correctly ad brought about ac tion. The direct proof that conformational changes did occur in the enzyme were also obtained by an extensio n of Gerhart and Schachman's studies on ATCase. Koshland changed this 'rigid' theory and proposed that the substrate and the enzyme were too flexible for such a theory. otherwise the change was detrimental for an optimal fit. separ ated by sedimentation were mixed again. In our discussion above we have seen that s edimentation coefficient depends very largely on the conformation of the particle. Proof forKoshland's Induced-Fit Theory Gerhart and Schachman's studies provided direct proof for another theory of extr eme.6 - . The theory proposed that the substrate and the enzyme acted like the key and the lock . the incorrect substrate could not fit likea bad key.332 Biophysical Chemistry could still bind CTP but had no catalytic ac. Figure 10. If the two subunits. If the correct enzyme-substrate approached each other the change was such that both com plemented each other. the molecule which was the same size as native ATCase and whic h had both the catalytic and the regulatory functions could be regenerated. 4. Succinate is a substrate analogue for this enzyme. i0-8 lO-S 10-4 10-s 10-i Succinate Concentrat/on (M) Figure 10. See the change tn the sedimentation coejclent of the enzyme as succate concentration increases. .3.6 1.8 0.0 1.8 2.2 0. the enzjme may become.2 = 2.4 2. Mark the antagonistic behaviour of CTP. more asymmetric as it binds to the substrate.26 Effect of succlnate (a substrate analogue) on the sedimentation coe . It tends to increase the sedi mentation coeffieenL Its opposite action here is reflected by the fact that it is an inhlbitor of the ATCase.L'lent of ATCase. Since the sedJmentatWn coefficient s decreasing.4. a boundary which sediments slowly is seen and the . Now there are two boundaries. temper ature. M is the meniscus and B signi fies bottom of the Subunits of Ribosomes . Thus ff we obs erve a at 7 S. However. NADH is a cofactor for the enzyme lactate dehydrogenase. which is an inhibitor for the enzyme. 10.333 The above studies were given even sounder footing when the two scientists demons trated that the changes in the sedimentation coefficient brought about by rising concen tration of the subtrate analogue could be reversed almost completely by adding CTP. (B} NADH bound to Idctate dehydrogenase . the two molecules sediment together. before and afte r binding. This is the theory behind studies of such bindings. {A] NADH alone sedim ents at 0. when chicken heart lactate dehydrogenase to the coenzyme solution a new boundary appears which has a sedimentation coeffi cient (Figure 10. concentrations of the enzyme and the cofactor themselves. can be observed by absorption optics ff the cofactor absorbs in the UV or the visibl e range. the cofactors have very small sedimentation coefficients. Binding of a Cofactor to an Enzyme Cofactors are tiny molecules as compared to the enzymes. This means that the cofactor will suddenly have a l arger sedimentation coefficient owing to this binding. NADH alone is sedimented.2 S and the other at 7 S. NADH absorbs well at 34 0 nm. The boundaries. However. This simple assay is one of the best tools to assay the binding of NADH to lacta te of course for other cofactors to their enzymes) as a function of such conditions ionic strength.27 Absorption plots of sedimentatlon of NADH (340 nm}. it must be that NADH is binding to the enzyme and settling with it. The enzyme does not absorb at 340 nm. As such. both absorbing at 340 nm : one sed imenting 0.sediments at 7 S. This is even more confirmation that NADH is bi nding the enzyme. when they bind to the larger enz yme.2 S peak diminishes.2 S.27). As t he of the enzyme is increased. the boundary at 7 S has more area under its peak area under 0. IfMg concentration is further reduced. . anothe r 8 and the third at 30 S (Figure 10. ff Mg÷ concentration the same preparation sediments giving three peaks: one at 70 S as before.28). co/t is sedimented at a high Mg÷ concentrati on. t he 70 S entirely and the 30 S and the 50 S peaks enclose more area. the giving a single peak sedimenting at 70 S. However. This tells us that t he ribosome is actually formed of two subunits that can associate and dissociate de pending concentration.If pure ribosomal preparation from E. However.28 Bfophgsfcal Chemistry Corlc. The sample is now m ixed in this concentrated heavy metal salt solution.334 'gure 10. of MgCl M 30S 50S 70S B Drawrgs of schl:leren paerns of rlbosome$ and subunlts sedimented at d{ferent co ncentrations of MgCl. It is almost mandatory here to use t he salts of heavy metals (cesium chloride. In the bottom rectangle the concentration of MgCl is ristrj from 0. What is done is that t he sample is initially mixed with the gradient medium to give a solution of uniform density.01 M MgCI. CsCI) or to use sucrose/mede. Since most of the principles applying to band sedimentation have been discussed earlier (preparati ve centrifugation} we will just try and see the problems and advantages of band sedimentation visa vis boundary sedimentation before we discuss the applications of this technique. Boundary sedimentation is more adapted to analytical centrifugaton. ironically. rib osomal subunits are fulb3 associated. 4. as the concentration rises. The gradient will then self-form when sedimentation is begun. At 0. This probl em. band sedimentation (density gradient centrffugation) differs significan tly from boundary sedimentation as we have seen while discussing preparative centrifugation. BAND SEDIMENTATION In the above section we discussed analytical applications of boundary sedimentat ion However.0002 M. When centrifugation starts. At this concentration. The top rectangle is at 0. some a ssociation into 70 $ particle is see. CsCI . be addressed by not preforming a density gradient at all. This is beca use it is difficult to prepare a density gradient in the small analytical ceil.0002 M. the subunits are completely dissociated. they ei ther rise or sink to the region where the solution density is equal to their own buoyant density. now reorient themselves according to their buoyant densities .molecules sediment to form a concentration gradient which automatically results in a densi ty gradient. Secondly. Once this happens. Since there is a density gradient. Another problem area in band sedimentation is the determination of sedimentation coefficient. But still it is used frequently as an analytical procedure to deter mine the buoyant density of a given particle. if the determination is being done in a p reparative . sample molecules which were previously uniformly distributed throughout the tube. the net force acting on the mole cule is never a simple function of distance. This technique suffers from the disadvantage that very long centrifugation times might be requi red to achieve equilibrium. there may be wall effects which would disallow a true value to be as signed to sedimentation coefficients. We know that the velocity of the particle increases as it moves farther from the centre of rotation.oefficients. this technique is the best to either separate them or study their behaviour. a diffe rent composition of gradient would be isokinetic. the rate of migration of the band will never be known. Thirdly. the movement of the band may not be visualized and therefore the investigator will have to de pend only ontwo valtes . there are its uses which m ake it an ensable technique. Also easy to see is the fact that if for a given centrif uge tube. ' While these problems do exist for band sedimentation. This detail can be ignored only at considerable pe ril to the experimental determination of sedimentation coefficient. The way around these problems is to determine relative value of sedimentat ion coefficient sedimenting the unknown particle with those particles whose seiimentation value has been determined by analytical centrifugation. again if a preparative centrifuge is being used. for a longer or shorter centrifuge tube. The second and the third problem cannot be addressed unless analytical dntrlfuge is used. The first problem can be solved by the use of isokinetic gradients. Bourdary . 5% to 20% sucrose gradient is isokinetic.the initial position and the position of the band at termination of exp eriment. If the mixture consists of particles with discrete sedimentat ion c. However. It is easy to see that t he concentration and viscosity of the gradient at any point depends on its distance from the cent re of rotation if it is to be isokinetic. Thus.Centrifugation 335 centrifuge. The concentration and the viscosity of the gradient at different points is then to be chosen so that this increment in the velocity is exactly nullified and the particle migrates constantly. The exact explanation of this is as follows. Isokinetic g radients are those gradients where the viscosity and concentration of the gradient at any poi nt in the tube is selected such that the particle migrates at a constant velocity at all distances from the centre of rotation. Such indiscriminate det erminations can and do quite often result in large scale discrepancies in the values ofs obt ained by different laboratories for the same particle. such values may frequently be far from the actual values. of double stranded Cs÷ DNA depends on its base composition: 0 = 1. p. if the mixture is heterogeneous. Single stranded DNA is -0. . RNA iis quite dense and CsC1 is not a suitable gradient maker for this nucleic acid. Thus gradients of Cs2SO 4 or sucrose are to be prepared. the RNA-DNA hybrids can be separated on the CsC1 gradients.sedimentation may fail here for the simple reason that if there is a minor compo neit of a high molecular mass among other components of smaller masses. Thus it is easy to separate these two with the help of density gradient cen trifugation.660 + 0. RNAs get separated on the basis of thei r size. the former may just bec ome indistinguishable.098X÷c Thus CsC1 gradient separates DNA on the basis of its base composition. the gradient is most often constructed with CsCI. Band sedimentation has been used extensively in the study of nucleic acid struct ure and function.cmL denser than the corresponding double strande d DNA. bound'y s edimentation is the best method to use. We give below a few examples. For DNA. The bu oyant density. On the hand.015 g. On the other hand. of Nucleic Acids Isopycnic ultracentrifugation is one of the most commonly used procedure s for separating nucleic acids. The stran ds higher GC content sediment faster. Role of Rho Factor in Termination of RNA Synthesis . Lysis is normally done with incubating the cells with lysozyme initially followed by a NaOH or SDSI treatment. the linear chromosomal DNA can intercalate a large amount of EtBr and unwind to a great extent. is nevertheless a great example of the us e sedimentation technique for the simple reason that it takes advantage of the sha pe of the macromolecules in order to separate them.336 Blph. will result in greatly reduced yield of purified plasmid. However. EtBr is an interca lating agent. ALSO notable is the fact that both boundary and band centrifugation are r esorted to. If lysis progresses well. As such it is hugely instructive of th e range of the technique and the manipulation that a researcher can resort to in order to explo it the technique fully. This is the trickiest part of the whole procedure because'both. the density of DNA goes on reducing. It binds to DNA by intercalating between base pairs. This leads to an interesting situ ation -. As inte rcalation proceeds. Ideal lysis is where the cell has bee n broken just enough to let the plasmid escape without too much of the chromosomal DNA coming out.the density of the linear DNA can reduce much more than the density of the plasmid. This makes the DNA unwind. This leaves a supematant that contains the plasmids. Isopycnic sedimentation is now carried out. As discussed above. incomplete lysis and total dissolution of cell s. A c ovalently closed circle such as the plasmid cannot unwind completely. most of the chromosomal DNA released is of high molecular weight and can be pelleted along with other cellular debris with high speed cent rifugation. CsCI is a fast substance and will generate a density gradient automatically. The first step in purifying a plasmid is the lysis of the host cell. At concentrations of EtBr. Th is in the densities of the two species of DNA leads them to band differently in the centrifuge lqading to their ultimate separation. The supernatant obtained above is then mixed in a solution of CsCI and ethidium bromide (EtBr). the plasmid has a higher density than the linear DNA. which is less used today.D sicctl. Chmtstvt9 Classical Method of Plasmid Purification This method. [4C] uridine triphosphate was one of the ribonucleotides and in the other UTP was present. the reaction mixtures from both the tubes wer e upon a preformed Isokinetic sucrose density gradient {59/0. . This is clearcut evidence that the [3H] RNA is shorter . After incubation. Rho factor was present. termination taking recourse to band sedimentati on. W. Roberts in 1969.We now know that Rho factor terminates RNA synthesis. The experimental setup had two reaction mixtures. The [3H] RNA is collected in a much lat er fraction compared to the [4C] RNA. RNA polymerase along with required buffers a nd conditions was present. In both. Figure 10. the E. presence of Rho has resulted in synthesis of shorter RNA meaning that it is inde ed factor. overall shape of the RNA molecules is the same. If Rho factor is indeed inv olved termination. sedimentation. In one of these. The behind the experimentation is simple and compulsive.29 tells us results of the sedimentation experiment. He isolated the protein fact or and that it had a role in synthesis. the tube was punctured and the fractions collected. In the one wher e Rho was not present.20%} and sedlmented. c oli DNA acted as the template. In both. This knowledge is based the experiments conducted by J. its presence in the reaction medium would lead to synthesis of smal ler RNA. smaller RNA would sedim ent slower bigger ones. . the sedimentation pattern of such a DNA will not vary in a normal density gradient. The strength of the hydrogen bonds and the hydrophobic associations wo uld not allow the two strands to separate.3 M NaOH. Sedimentation in denaturing gradient has been a variation highly used in nucleic acid studies. On the contrary.. This seemingly simple event induces large scale changes in the sedimentation behaviour of DNA and allows stu dy of phenomena which could not be studied in normal density gradients. . Denaturing den sity gradients mean isokinetic gradients (5% . And if there are more breaks tha n one.. The main uses of this variation are given below.29 Effect of Rho factor on sedlmenmtion rate of the sIntheslzed RNA.. while the dashed line represents RNA made with [C} LrI'P.. [5 10 15 20 Fraction No. .. the two strands would separate and the strand in which the break exists will migrate as two different fragments..30). Th e solid line represents RNA made with [H] UTP.se many fragments will be created and all will migrate independently if their base composition is sufficiently different or if they have sutT1ciently different sizes. if such a DNA is migrated in an alkaline gradient {Figure 10.' '. First of all one must consider the fat that if a single strand break occurs in a double stranded DNA.20% sucrose) in 0./ l. Denaturing pH lgure 10.30 Denaturation of DNA ges rise to derent specles of DNA for the intact .. thoo. ljure I0..1 to 0.337 Centrifugation . DNA denatures and the two strands separate. At high pH. and the nP. " .kc. d double-stranded DNA. l for y generaons a mium coning of e ces con e bel at r place. If s DNA ent. a s con of e DNA dents we slowly obaon tes of DNA at Is double sd. repcag DNA must be se sd as sin fraents which must later be veny by e ee gase. Hoer. dloac c repcag poon of DNA. ese aents e ed Ok frents er e ese conclusions have been cated d today we ow at DNA ds pte d/scont/nuous manner. Native Denatured The same logic that has gone into the above study can also let us know about les s thLngs such as the rate of depur/nat/on (depurlnSon does not lead to s/ngle stra nd .cared onuoy If one o E. d such corated raoac dents at a low s. most DNA sedenm a d hang a we s.338 Bophyscal This may seem qu/te/nslgniflcant actually. D Re. at s at aut dg out bs DNA? Nog cept at s s-e we se bs wch was lot at DNA reptes a dsconuous mer lea g to e pucon of what e O fraents (s chapter 13: 'pulse-chase labeg'). e conclusion ached Is at e se stud bs e DNA. when s ted e adlen. non-radloacve mi e pulse ladled [H] e DNA sedent e aent. ere Is oy one laon for Is. a we low s. If E. But one couples e and obaon e t. However. different conformations a ssume widely differing s values making the identification of these conformations more easily possible. How to Confirm that a Given DNA is a Covalently Closed Circle If one grows bacteria containing a plasmid in a medium containing [3HI thymidine . The d isappearanc .Centrlfugation 339 however. and radiation damage to DNA. and the other having a large s value which could be due to a closed circle. all DNA is labeled. with such drastic change upon denaturation. each depurination will lead to single strand br eak and can be identified as such). Thus a linear molecule and a nicked circle do not hav e widely different s values. a nicked circle and a n on-supercoiled covalent circle have the same s values.31 will make evident the effect of denaturation on some of the conform ations of DNA.one corresponding to the bacterial chromosome ( whose s value is considerably smaller owing to the fact the gradient is denaturing and t he strands would have separated) . It is naturally difficult to distinguish between these conformations of DNA by conventional density gradients. even where there are differenc es. The peak area tells us that the rapidly sedimenting band corresponds to a very small fraction of the total DNA. denaturing gradients are of considerable value here. Linear DNA and cova lenfly cosed circular DNA may differ from each other in their s values substantially. This will ma ke the resulting structure to sediment slowly and the fast sedimenting band will disappear. these may not be sufficiently large. How to confirm that it is indeed a closed circle? A look at Figure 10. If these cells are now lysed and the DNA sedimented through an a lkaline gradient. they give rise to an open circle and in a denaturing gradient the strands will separate. There is another use for denaturing gradient in DNA studies. Naturally. the loss of a purine base exposes the phosphodiester bond there to alka line hydrolysis.31 points a way out if closed circles are nicked. Moreover. in alkaline gradients therefore. This data merely indicates that the band could be a c losed circle. two bands are observed . A look at Figure 10. Likewise a nicked circle anda naturally occurring supercoil have close ly situated s values. B ut this is not the case with all the conformations of DNA. For example. the approach to equilibrium method is also used. Electrical forces. I M KCI. 5..When the ultracentrifuge is operated at high speeds.w D(1-p) . A third method. like dou ble layer phenomena (see chapter on colloids) will produce an ion cloud which can impede the movemen t of large It is therefore ncessary to use either isoelectric protein or to add a neutral s alt like 0. DETERMINATION OF MOLECULAR WEIGHTS Two approaches are in vogue for determination of molecular weights through centr lfugation. the randomly dist ributed particles migrate through the solvent radially outwards from the centre of rotation.or sedlmenting particle can be determined by the Swdberg equation RTS0.is confirmation that the fast sedimenting band was indeed a closed circle. the former being more popular. The molecular weight of macromolecule. The pH or ionic strength is adjusted to make the macromolecules neut ral. the sedimentation velocity method and the sedimentation equilibrium method. The i nformation hbout formation and velocity of the boundary between the portion of the solvent that has cleared of macromolecules and the portion of the solvent still containing the macromolec ules is provided by either Schlleren or Rayleigh interference optics. (i) Sedimentation velocity : The method measures the speed of the moving boundar y (represented by a peak due to increase in refraction in the region where macromo lecules are present). density of the medium. partial specific volume of the macromolecule. absolute temperature. Biophysical Chemistry . sedimentation coefficient of the molecule (see below). gas constant. diffusion coefficient of the molecule.340 where anhydrous molecular weight of the macromolecules. T = absolute temperature. The concentration gradient set up in the analytical cell can then be measured and substitution of the values in the following equation gives an idea about the molecular weight of the macromolecule where R = gas constant. with short analytical cells (I-3 mm) and use of overspeeding techniques the time has been shortened to a few hours.Centrifugation 341 {i Sedinetttbn et/tilibrtum : This is a method which does not require measuremen t of diffusion coefficient of the macrom01ecule whose molecular weight is to be calcu lated.The technique in its present form is more popular tha n the sedimentation velocity technique. = partial specific volume.2 ght by Sedimentation umbr|um : Some Problems subjectedt . The method involves centrifuging a macromolecular solution at a spe ed which will exactly balance the tendendy of the macromolecule to disperse in the opposite di rection due to diffusion. p = density of the solvent. Ho wever. . In this respect it scores more than the sedimentation velocity method for which diffusio n coefficient is a necessity. o = angular velocity. Box 10. The centrifugation is continued till a balance is established between the sedimentation and diffusion of the macromolecule so that no net movement occurs any more. C and C2 = concentrations of solute at distances r and r2. Obtaining equilibrium used to take a very long time (several days to a week). since it depends upon the data from the extremities of the solution column. . Thus. since the revival of sedimentation equilibrium technique with specially designed small and narrow cells and better suited optics (Rayleigh interference). two places exist where flow of solute is zero. Initially this method had the advantage of rapidity over the conventional sedime ntation equilibrium method.. However.342 Biophysicat Chemistry (ill) Approach to equilibrium: Calculation of molecular weight by sedimentation equilibrium method depends upon the fact that when equilibrium is reached the flow of solute vanishes at every point in the cell. However. even without centrffugation. Quite some time is required for the tube to reach such an equilibrium through continued centrifu gation.e. the equation for sedimentation equilibrium must apply to these points at any tim e from the beginning of the experiment. Moreover. This particular application of sedimentation equili brium method is known as the Archibald method (Archibald pointed out the above possibility). This stateme nt Is based upon the fact that matter can not be pushed through eithe of the extremities. i. precise measurements are not possible and the method is inferior in accu racy. These places are the meniscus of the solution and the bottom of the cell. there is not change in concentration at any point . at all time. f or all purposes. this method has m ore or less fallen into disuse. when he migrates another aliquot on an alkaline gradient he finds that apart fro m a major band.Centrifugation 343 Exercise I.000. State whether true or false. What may have happened to the DNA? 6. He remembers that the pH of" the solution was somewhere near 4. Can you provide an explanation? 2. 3. 4. However.000 rpm would take 15 minutes to reach the same position when the rpm is doub led to 20. Give reasons for your answer. However. upon sedimenting you find that they sediment at di fferent " rates. Later in the day he switches his mind and decides to sediment a protein solution. there are several other bands that migrate slower. He uses the same gradient. A worker left a DNA solution on his table for a week. In order to detect whether the DNA is still alrig ht he sediments a small aliquot on a sucrose gradient and finds that a single band is observable . 5. You have been given two DNA solutions and told that they have identical base pair composition and the same length. Is this statement true or false? Provide math6matical proof for your ans wer. A boundary which takes 30 minutes to reach halfway down the cell when ce ntrifuged at 10. What kind of sedimentation behaviour is expected of a protein that becom . You are given a solution of DNA and told to prove that it contains doubl e stranded DNA. Can you explain why?. We believe he is in a spot of trouble.000 rpm will take 60 minutes to reach the bottom of the cell if the rpm remains cons tant. In how may ways can you prove this fact? 7. A boundary that takes 30 minutes to reach halfway down the cell when cen trifuged at 10. A student prepares an isokinetic gradient for sedimenting DNA. Academic Press. the worker finds out that none of the regions in the tube absorbs at 260 nm. 2. The Ultracentrifuge. . (1959). (1940). again a single boundary is observed. A protein Solution that has been purified sediments as a single boundary . After treatment with PMB. D.O. K. However. Schachman.D. London. G. New York. Centrifugal Separations in Molecul ar and Cell Biology. Butterworth.K. Ultracentrifugation in Biochemistry. (i 970). How do you explain this observation? 9. Why should that be so? Suggestions For Further Reading I. Lond on. Bowen. Birnie. Oxford Universit y Press. (1978). T. H.es more compact after denaturation? 8. An Introduction to Ultracentrifugation. After conducting rate zonal sediment=tion of RNA in a CsCI gradient. T. the boundary migrates ve ry slow compared to the earlier boundary. and Rickwood. Svedberg. A particular sedimentation run takes 40 minutes to complete. 4. John-Wiley. 3. New York.J. How can you complete the run within that time? 10. yo u have to leave the lab after 20/25 minutes. However. and Pederson. The feature common to them all is that two mutually Immiscib le phases are brought into contact with each other. be achieved readily using chromato graphy. most amino acids resem ble one another rather closely in physico-chemical properties. A separation might. Thus the name of the process was coined from the Greek words for color (chromo) and " to write" (graphy). the mobile phase either moves oxer the surface or percolates through the interstices of the stationary phase. a Russian biochemist. The component showing least interaction with the mobile phase whi le interacting strongly with the stationary phase migrates slowly (retarded).11 CHROMATOGRAPHY If the individual components of a mixture have widely dissimilar physical and ch emical properties. whi le the other Is mobile. But as the individual components of a mixture get more and more similar in physical and chemical properties. For example. The first detailed description of chromatography is generally credited to Michae l Tswett. It is impossible to separ ate a given amino acid from a mixture of several by conventional separation methods such as fracti onal crystallization. One of these phases is stationary. .whi le being carried through the system by the mobile phase. Since these different rates of interactions govern the migration of the sample components through the system. The compound which interacts more with the mobile phase and least with the stationar y phase migrates fast. however. who separated chlorophyll from a mixture of plant pigments i n 1906. it is very easy to separate one from another. Because of the nature of the pigments in the sample. The term chromatography bunches together a family of closely related extremely p owerful separation methods. it become s increasingly difficult to separate them from one another. each band had a distinctive color. each one of the components migrates at a differen t rate. Different components of the sample mixtu re interact with the two phases differentially on the basis of small differences in their ph ysico-chemical properties. This differential movement of the components is responsible for their ultimate separation from eac h other. The sample mixture. introduced into the mobile phase under goes repeated interactions (partitions) between the stationary and mobile phases . immediately distributes itself between the st ationary and the mobile phase. If. the stationary and the mobile phase. which is expressed as follows . the mobile phase fl ow is stopped. the compound will be in equilibrium between the stationary phase and the stopped mobile phase. the concentration of the compound in each of the phases is described by the partition coefficient. at a given time during chromatography. As described above. the solute upon entering a chromatographic system. K. At this stage.Partition Coefficient Partition coefficient {also known as distribution coefficient) is a definitive t erm normally used to describe the way in which a given compound distributes or partitions its elf between two immiscible phases. liquid. the correct distribution coefficient will be give n when one considers the activities of the compound in the stationary and the mobile phases rather th an their Concentrations. covalent. reflects the relative attraction or repulsion that the molecules of t he two show for the solute molecules and for themselves. reversible interactions importance.se {stationary phase) and carbon tetrachloride (mobile phase) is 0. co-ordination). The partition therefore. Dispersion forces and electrosta tic forces which contribute most to partitioning of the solute between Let us first deal with the dispersion (London) interaction. The choice of the mobile and stationary phase is made in s uch a way that components of the sample to be resolved have widely differering partition coeffi cients. some chemical reactions can not be excluded from It is the molecular constitution.Chromatography 345 where C and Cm are the concentrations of the compound in the stationary and the mobile phases respectively (actually. This interaction is of a non-polar . The mobile phase may be liquid or gaseous. concentration is used instead of activity). although. Types of Interaction: The Nature of Partition Forces The distribution of a solute between the stationary and the mobile phase is a re sult of the between the solute molecules and the molecules of each phase. These attractive or repulsive accompanied by a release or consumption of energy.2. In chromatography. which is fixed by the species of atoms present and by the of bonds between them (metallic. All chromatographic systems consist of the two phases named above. The stationar y phase may be solid. which decides the and the intensity of the physical interactions. ionic. but since there is no way of determining the activitie in a chro matographic system. The amount of interaction provides a measure of the strength of the interaction and serves as a criterion to classify physical or chemical in nature. most of the interactions that are of interest in chromatography are physical in nature. gel. or a solid/liquid mixture. This concept of partition coefficient is the basic principle of all chromatograp hic methods. Thus. which is immobilized. it means that the concentration of the substance in carbon tetrachloride is five times th at in cellulose. if the partition coeff icient of a substance between cellulo. it is easy to understand that a solute molecule will interact more with the phase which is non-polar. Thus. . Let us now deal with the polar interactions. These non-polar molecules do not posse ss any dipole moment. this solut e move fast if the non-polar phase is the mobile phase or will be retarded more an d if the non-polar phase is the stationary phase. the weak attraction changes to' repulsion. The interaction is a resultant of instantaneous dipoles formed the nuclei and electrons at zero-point motion of the molecule. Another nonpolar (whether a solute or a solvent) will mix in all.In a non-polar liquid such as carbon tetrachloride. non-polar exist in a state of random distribution to give a disordered array. a polar sample component will interact more with the phase which polar and move fast or be retarded more depending on whether the polar phase is the or the stationary phase respectively. Thus only thos e solute which exert either a higher or at least equal attraction with the solvent molecu les as to the attraction of solvent molecules for each other. The solvent whose molecules have permanent dipoles exhibits much intermolecular attraction as compared to the non-polar molecules.proportions since neither kind o f the any attraction between them. will be able to mix with the Therefore. From the foregoing. Dispersion forces are When two non-polar molecules of the same type approach each other closely enough for orbitals to overlap. The first kind to be discussed is t he interaction. London's dispersion interact ion is only force present between two molecules. Yet another interaction important for a comparatively r ecenfl developed chromatographic procedure is the interaction dependent upon biological specifici ty between a macromolecule and a ligand (affinity chromatography. structure. If solute molecules have to inter act with such a polar solvent. they need to break this highly ordered. as all chromatographic procedure s involve differential distribution of solute molecules between two phases. However. or it should have the ability to ionJze. see later). important in chromatography. In addition to above interactions. which can be grouped as physical in nature. Hydrogen bonding influence s partition of solutes and therefore their chromatographic separation tremendously when one of the phases has hydrogen-bonding ability. Roughly 4-6 Kc al is required to break each hydrogen bond. A common example of a solvent which is stabilized by hydrogen bon ding is afforded by water. resonance. The actual strength of hydrogen bonds varies with d ifferences in molecular geometry. oxygen. Yet. Those solute molecules which have either of these abilities interact maximally with polar solvent like water. t here are other interactions. These forces do not fall under the perview of true partition as in actual terms partition concerns itself with differential solubility of a solute into two knuniscible solvents. . The one interaction of this type which is extremely important for ion-exchange c hromatography (see later) is the ion-ion interaction characterized by electrostatic attraction between mutually oppositely charged long. that may be grouped as chemical in nature. a generalized statement may be made: the interactions arising due to hy drogen bonding are relatively strong and in some instances interaction energy may approach that of a weak chemical bond. relatively ope n structure consisting of clusters of 4-5 water molecules. It exists as a highly hydrogen bonded. and the nature of the neighbo ring atoms. To do so. all of the forces involved may be discussed as partition forces. albeit.. Othe r non-polar solute molecules simply do not interact with polar solvents. acld-base behavior. nitrogen. the solute should either have the ability to form hydrogen bond with water.g.346 Biophysical Chemistry Another type of polar interaction which is very important In chromatography Is hydrogen bonding. A hydrogen bond is formed between a molecule in which hydrogen is attached to strongly electronegative atoms (e. fluorine) and a molec ule which possesses unshared pairs of electrons. in which many repetitive partition steps take pla ce (see Box 11.C.1). The Partition Principle: Partition Chromatography When a solute is allowed to equilibrate Itself between two equal volumes of two immiscible liquids. the ratio of the concentration of the solute in the two phases at equil ibrium at a given temperature is called the partition coefficient.SURVEY OF CHROMATOGRAPHIC PROCEDURES Each of the chromatographic procedure described below utilizes different types o f interactions to achieve resolution of sample components. first developed by L. A mixture of substances with di fferent partition coefficients can be quantitatively separated by a technique known as countercurr ent distribution. Craig. . 347 Chromatography . though nert. Martin and R.348 Biophysical Partition chromatography is a logical extension of the countercurrent partition principle for achieving chromatographic separation of mixtures. This tightly bound water. or the granules of cellulose in paper.P. The separation is achieved in a hge number of partition steps which takes place on microscopic granules of a hydrated insoluble inert substance. The solute molecules are subjected to microscopic partition processes between the immobilized water layer and the flowing solvent. The gr anules. the only factor which Influences the movement of a compound as the solvent travels along the stationary phase is the relative solub ility of that .L. The principle of partition is exploited in gas/liquid chromatography (GLC) techn ique also. originallyd eveloped by A. The technique. since it is immobilized. Synge has since been applied to an enormous nunber of s eparations. such as starch or silica gel packed in a column or layered on a plate. Separations depend upon the partition of the solute molecules between a li quid.M. are hydrophilic and as such are surrounded by a layer of tightly bound wat er. Since the process takes place o n the surface of each granule the number of partition steps is so great that the substances move along the column or surface at a different rate as the mobile phase flows through it. supported on a suitable solid. and the gas flowing through the system.J. On the sur face of this stationary phase flows the mobile phase of an immiscible solvent containing the mixture to be separated. In true partition chromatography. serves as the stationary phase. Adsorption is a surface phenomenon which signifies a higher concentration at an interface as compared to that present in the surrounding med ium (see chapter 7). Adsorption should not be confused with absorption. Ion-Exchange Chromatography This procedure was first developed by W. The ion exchanger consists of an inert support medium coupled covalently to positive (anion exchanger) or negative (cation exchanger) functional groups. The solute molecule which interacts more with the adsorbent. Adsorption Chromatography Substances differ in their adsorption-desorption behaviour between a moving solv ent (a liquid or a gas) and a stationary solid phase. if anion exchange chromatography is performed. This behaviour of a substance can be exploited to achieve its separation.Chromatography 349 compound in the two phases. which signifies t he penetration of one substance into the body of another. T hese sample long will be retarded whereas other uncharged or positively charged long will no t be retarded to the same degree and will be eluted out fast. Substances more soluble in the mobile phase will mig rate greater distances as compared with the substances more soluble in the stationary phase. Cohn and may be defmed as the reversibl e exchange of long in solution with long electrostatically bound to some sort of i nsoluble support medium. Thus. In this way a separation of sample components is achieved. it u sually denotes interactions involving hydrogen bonding and weaker electrostatic forces of the substance with the adsorbent. which is also the stationary phase. For the purpose of chromatography. through electrostatic attraction. The situation will be exactly rever . Other compounds of intermediate solubility between the two phases will migrate to inte rmediate distances depending upon their partition coefficient. To these cov alently bound functional groups are bound. the term adsorption has limited meaning. negatively charged sample components will interact more with the s tationary phase and will be exchanged for like charged long already bound to the matrix. is retarded more while less interacting solute molecu les are retarded less. oppositely charge d long which will be exchanged with like charged long in the sample. sed for cation exchange chromatography. The suppo rt medium. . This technique is also used to determine relative molecular weight of a given macromolecule. Molecular Size: Gel-Filtration Chromatography This technique exploits the molecular size as the basis of separation. consists of porous beads where pore size is strictly controlled. The enzyme which is specific for the substrate analogue binds to the gel becoming immobile while all other components move down and out..high resolution power. this is ultimately the basis of separation also. Affinity Chromatography The technique utilizes the speciicity of an enzyme for its substrate (also recep tor for its agonist. Figure 11. a gel. antibody for antigen) or substrate analogue for the enzyme's (other pro teins with biological specificity) separation. Macromol ecules smaller than the pores get entrapped in the pores (and move slowly).1 provides an overview of chromatographic procedures on the basis of t he stationary and mobile phases used. while those bigger than the pores travel unhindered through the column (and elute out faster than the smaller mole cules). The technique has a very. Thus the main interaction between the solute and the stationary phase is with respect to the size and . A substrate analogue is coupled to the gel matrix and t he cellular suspension is allowed to percolate through. ANAR NAME OF CHROMATOGRAPHIC METHOD GAS LIQUID CHROMATOGRAPHY (GLC) .LIQUID BONDED LIQUID SOLID PLAIV COLuMN PLANAR NATURE OF NUMBER OF MOBILE PHASE STATIONARY PHASE GAS [-.LIQUID CHROMATOGRAPHY --SOLID SEPARATION PHENOMENA PARTITION -AI)SORFI'ION -PARTITION TECHNIQUE COLUMN -COLUMN -COLUMN -PI.-. ADSORPTION HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPTCL) HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY (HPLC) LIQUID SOLID CHROMATOGRAPHY (LSC) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) THIN LAYER CHROMATOGRAPHY fI'LC) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) PAPER CHROMATOGRAPHY (PC) ION EXCHANGE CHROMATOGRAPHY (IEC) "-.ION EXCHANGE -THIN LAYER ION EXCHANGE CHROMATOGRAPHY (TLC) PAPER IRON EXCHANGE CHROMATOGRAPHY -.EXCLUSION COLUMN PLANAR .GAS SOLID CHROMATOGRAPHY [GSC) CLASSISCAL LIQUID LIQUID CHROMATOGRAPHY (LLC) PAPER CHROMATOGRAPHY (PC) CHROMATOGRAPHY CHROMATOGRAPHY -COLUMN MODIFIED PARTITION -PLANAR -coLuMN --. .GEL PERMEATION CHROMATOGRAPHY (GPC) THIN LAYER GEL PERMEATION CHROMATOGRAPHY (TLGPC) -.AFFINITY COLUMN AFFINITY CHROMATOGRAPHY Figure 11. I Class]Icatlon of chromatographic systems. However.Chromatography 351 TECHNIQUES OF CHROMATOGRAPHY There are two basic techniques of chromatography: plane chromatography and column chromatography. the p ioneer of liquid partition and gas chromatography. adsorption chromatograph y can be carried out either on a column or on a thin layer. Apart from its obvious simplicity. A. the same is true for partitio n and most other types of chromatography. Ancient Romans used this technique to analyse dyes and pigments. I. Each of these technique s have their specific advantages. A "thin layer" of adsorbent spread on a glass plate is used much in the same way as a piece of pap er. chromatography on plane surfaces. PLANE CHROMATOGRAPHY A drop of liquid spotted on to a piece of paper or cloth will spread in a circul ar pattern. are types of chromatography (based on different principles) which can be carried out using any of the techniques stated in the preceding paragraph. plane chromatography techniques require only minute amounts of samples. will be fruitful for understanding various types of chromatography. A discussion of the techniques. Within a few y ears of Martin's publication. the stationary pha se in column chromatography is packed into a glass or plastic column. applications. concentric rings of colors will be observed. ion-exchange chromatography etc. There are two variations of plane chromatography: paper chromatography and thin layer chromatography. partition chromatography.. In thin layer chromatography the stationary phase is coated onto a glass or plastic surface. It is important to distinguish between types and techniques of chromatography. In more recent times. Moreover. . thus. rather than with a column. the modem day scientist re alised the value of paper chromatography only in the very recent past. this technique has helped to revolutionize biochemical research. If the liquid possesses color. In plane chromatography the stationary phase is coated on to a plane surface. Stahl popularised the concept of thin layer chromatography (TLC). In paper chromatography the stationary phase is supported by cellulose fibres of the paper sheet.P. Thus.J. As opposed to plane chromatography. and modes of operation. Adsorption chromatography. Martin. described paper partition chromatography in wh ich moisture clustered around cellulose fibres served as the stationary phase. This gives better results. Cellulose fibres in the paper hold moist ure tightly through formation of hydrogen bonds. A. etc. which gets deposited on the paper while it is being processed. The oxidation products usually are aldehyde. While these modifications lead to different mechanisms of separation . however. . ketone or carboxyl fun ctional gr0ups. This operation exploit s selective properties of two different solvents in developing a single chromatogram. silica gel.offers the unique advantage of two-dimensional operation. contains several thousand anhydro-glucose units linked th rough oxygen atoms. T hese impurities may be removed by washing the paper with 0. Many of the hydroxyl groups of glucose. a homo polysaccharide of glucose. PAPER CHROMATOGRAPHY Nature of the Paper The paper commonly used consists of highly purified cellulose. The cellulose itself takes a negative charge in com pany of water. The paper exhibits weak ion exchange and adsorptive properties. become partially oxidize d during manufacture. adsorbed salts and mineral matter. Modified forms o f paper have been produced in which the paper has been impregnated with alumina. ion exchange resin. the technique remains the same.1 N HCI and drying it before chromato graphy is carried out. Cellulose.The paper also contains impurities through inorganic substances. 2 is good enough for simple chromatography. which may be employed for the development of pape r chromatograms .2 Methods of paper chromatography. Of these. A micropipette can also be reused after its tip has been disposed an d a new tip applied.2).ascend/ng or descending techniques (Figure 11. After equilibration of the chamber is achieved.. Any device. The apparatus illustrated in Figure 11. This is done before dipping the paper into the eluting solvent. or a mic ropipette. There are two main techniques. In both cases the solvent is placed in the base of a sealed tank or glass Jar to allow the chamber to beco me saturated with the solvent vapour. platinum wire is preferred because it can be reused with several substanc es after heating on a flame.352 Biophysical Chemistry Apparatus and Paper Development The apparatus required for paper chromatography consists of a support for paper. Generally used devices are platinum loop. For some methods the sample may be applied as a narrow streak at right angles to the flow of the solvent. a solvent trough. (A) Descending. which can transfer a small volume of sampl e. (B) Ascending The sample is applied to the paper as a small spot. C "rrou wlO developing solvent' Cllp-per -. capillary tube. and an airtight chamber in which the chromatogram is developed. Equilibrating solvent M/grating sample Solvent Figure 11. can be used for spotting. the developm ent of the . In the descending techni que. the resolution of sample by ascending technique is somewhat b etter as compared to the descending technique. The disadvantage of the as cending technique. This is so because in ascending chromatography. The descending technique. Under the influence of these two forces. Secondly. Ascending technique has two advantages. the paper is allowed to hang in'or is suspended in a manner that the base of the paper is in contact with the solvent at the base of the chamber: The sample spots should be in a position Just owe the surface of the solvent so that as th solvent moves vertically up th e paper by capillary action.chromatogram may be started. two fo rces are acting on the solute: the capillary force. Firefly. separation of the sample is achieved. Separation of the sample is achieved as the solvent moves downward under gravity. the set up required for is very simple. Development is started by adding the solvent to the trough. If the development is to be performed by the ascend ing technique. however. is that it is very slow. the sample components are resolved better than in the descending technique. which makes it move up. the end of the paper near which the samples are located is held in a trough at the top of t he tank and the rest of the paper allowed to hang vertically but not in contact with the solvent in the base of the tank. and the gravi tational force which opposes this movement. on the ot her hand is . much faster than the ascending technique. one can choose the techlque which sts one's p'-pose most. If the components to be separated are coled. of Solvent System Usually in paper chromatography the stationary phase is water since it is very w ell adsorbed cellulose. less used techrque is the . is developed la the normal fashion by either ascending or descending procedure. Alternatively. a lar ge circular glass jar covered with a glass plate serves as a very good chamber. (B) Second development in a direction at rtjht anc jles to the. Thus. The resolution of components by tls techrque is sharper. One way is to press the pap6r between two glass plates with a ho le in the center through which the wick can be connected for solvent supply. This paper is then turn ed 900 and developed a econd time with another solvent having totally different eluting pro pertiesl Since the two solvents used have different eluting properties. In this method the sample is spotted at the center ofa crcularly cut disc of paper wlch is placed h orzonty. The center of the paper is connected with a wckto the solvent. (A} First development in the direction indicated b the arrow does not resolve B and C mpletely.techrque of radkl development.first using a different solvent system resolves all components completely. not be necessarily immiscible with water if water is being used as the station ary . The developme nt is continued until the faster moving component or solvent front approaches the. components which could not be separated using one alone can be easily separated by this procedure as shown is Figure 11. The paper. which is less polar flows over the polar stationary phase The . the chromatogram developed by t-s meth od looks pleasing to the eye. Two-dimensional chromatography. the distribution coeffi cients of individual also differ. wlch is placed at the base of ajar. Based On the above advantages.3 (also se e Box This technique is known as two-dimenslonal chromatography. with the sample applied as a spot close to a corne r. A thud. end of the pa per. The solvent rses up the wick and thence onto the paper through capillary action. The smple components now move outward radially formg concentric circles of increasing diam eters. A plane surface is amenable to sequential development in two directions using tw o differentsolvents. The paper is then removed and the solvent is allowed to evaporate. The mobile phase. Th e apparatus also is simple. esters. which must be considered before of a solvent system"is made One important consideration is to limit the number o f in the solvent system used to the barest minimum. apart from optimum separation.is because the stationary phase water i very tightly held by cellulose and will not the mobile phase on this account. phenols. There are other factors. amines. and hydrocarbons etc. The mobile phase is usually a mixture of vario us such as alcohols. This is so since the more . The are selected in such a way that the resolution of sample components is satisfact ory. ketones. acids. Ideally. It is necessary to add metal-chelating agents to these solvents be fore they ar used. The temperature. ('i) The solvent should be stable. Such a' choice would lead to maximum separation. The components of the solvent system should be so cho sen that the extent of evaporation of each individual component is more or less similar. It should not become oxidized when spread ove r paper. Desirable characteristics that a solvent should possess are listed below. the sample components should have differing solubilities in the two ph ases. nust be maintained within strict limits during the entire experiment.354 Biophysical Chemistry components a solvent system contains. Some solvents. In this case it becomes necessary to remove al l traces 0 the first solvent since even small amounts of the first solvent remaining on the paper will change the characteristics of the second solvent and will therefore interfere wi th the separation process. the solvent system should be so chosen that the two phases are immiscib le. of the separated zones will also be milxtmal. This material then migrates with the solvent front and dis torts the chromatogram. react with small quantities of copper present in the paper and get oxidized to p roduce brown of black tarry material. (//) The solvent should be removable from the paper and to this effect its boili ng point should ideally be less than 200°C. Differing extent of evaporation of the solvent components can change the composition of the solve nt and can lead to serious anomalies of separation. The time required will also be short an d the spreadlng. The temperature chosen for development should also be decided after serious deliberation because individual components of the solve nt will differ in their extent of evaporation at different temperatures. Moreover. for example phenolic solvents (particularly in ammoniaca l environment). (/) Partition coefficient of substances to be analyzed should range from 1-100 i n favouro the aqueous phase. the more difficult it will be to maintain a saturated atmosphere in the chamber. A few solvent systems used to separate diverse compounds through paper chromatog raphy . there fore. This property becomes all the more essential ff two-dimensional chromatographyis to be performed. If the sample components are c olored.I Examples of Solvents System Used in Paper Chromatography Solvent systems (/) Ratio Compounds Butan. they can be imparted color by spray ing the paper .1-ol/acetic acid/water Butan.1-ol/pyridine/water Propan.1-ol/pyridine/water Methanol/pyridine/water Butan. the analysis becomes simple as the distinctive color itself identifies the compo nent.1-ol/petroleum ether Chloroform/petroleum ether (40/10/5 (33/33/33) (25/12/63) (50/28/22) (4/96) (30/70) Amino acids Amino acids Amino acids Mono and disaccharides Plant pigments Plant pigments Detection There are various methods of detection available.1 Table 11.are given in Table 11. When the components are colorless (usually they are). ACID HYDRINDANTIN ¸O O II II .C\ 3H:O II O OPURPLE PIGMENT Chromatography 355 with color producing reagents. NH. 0 OH OH O NINHYDRIN H l " .COOH *" -. OH + R-. A case In point is the detection of amino acids. II AMINO 0 O .C-. Ninhydrin reagent spread on the paper reacts with amines and amino acids to form a blue or purple color. The identification of a given compound may be made on the basis of the distance traversed by the solute relative to the distance moved by the solvent front. by R for convenience. and (//0 radioactivit y. . the reagents may be applied to the paper by either imme rsing the paper in the reagent or by treating the paper to the reagent vapours./.2) the distance moved by the solvent = Rf the distance moved by the solvent front In case of carbohydrates. and is constant for a given compound under standard co nditions (see Box 11. this value can be exploited to de tect the unknown compound by matching its retardation factor value to those of the known compounds. the distance moved by a carbohydrate the distance moved by glucose = R° Since each compound has specific R (Ru) value. which reflects the distribution coefficient of the given solute. Other methods of detection are (0 ultraviolet and Infrared absorption. (//) fluorescence. G stands for glucose. Thi s ratio. If not sprayed. the components may be extracted and chemical and physical tests be performed on the extract. Otherwise.The spots due to amino acids can now be detected on account of the blue color th at they develop. is known as the retar dation factor (also known as re/at/veJ/ow). the term R is replaced. the layer can be dried and the components detected by various metho ds available.AppHeations The technique of paper chromatography has revolutionized biochemistry where diff icult analyses with vanlshingly small sample volumes are legion. Preparation of the Layer The glass plate on which the thin layer is prepared should be even and is thorou ghly -washed and dried before layer application. the study of ripening and fermentation. The material of which the thin layer is to be made (silica gel. More recently. To briefly describe the technique. In this case. the analyses of cosmetics. This slurry is ap plied to aplate surface as a uniform thin layer by means of a plate "spreader" starting at one e nd of the.2) is usually mixed with water in such a proportion that a thick suspension.. Kieselguhr. The sample to be separated is spotted at one end. or alumina) or by the partition principle (ff the layer is prepared by a substance such as silica gel which hold s water like the paper). and to top it all. a thin layer of a freely divided substance is deposited on to a flat glass plate. known as slurry results. thin layer gel filtration chromatography is also performe d as we will see later. The control of purity of pharmaceuticals. for more details see Table ii. THIN LAYER CHROMATOGRAPHY This technique is more or less similar to paper chromatography as far as most of the operations involved are concerned. After the development.plate and moving to the other in an unbroken uniform motion. Thus. the analyses of the reaction mixtures in biochemical labs are all performed routinely with paper chromatography technique. the layer is made up of controlled pore beads of various ge ls. most of the previous discussion applies here. The pla te is dipped into the solvent in a glass jar and the development carried out by the ascending tech nique. the detection of drugs and dopes in animals and hu mans. etc. B. Thin layer chromatography may be either carried out by the adsorption principle (if the thin layer is prepared by an adsorbent such as Kieselguhr. Modern thin layer chromatography kit s provide plastic or foil plates in lleu of a glass plate. The nature of the desired . the detection of adulterants and contaminants in foods and drin ks. 25 mm. for analytical separations the thickness of the layer is usually 0. gel . Thus . while for preparative separations the thickness of the layer might be about 5 mm. layer technique can be used for many different types of chromatographic separations such as adsorpti on. Although thn.chromatographic separation dictates the thickness of the slurry layer used. Silica Gel G Silica Gel G Silica Gel G Ethyl acetate/propan-1-oi (65/35) (Butan.Kieselguhr G (sodium acetate] Kieselghur G (sodium Phosphate.. it is usually adsorption type which is most used with this technique.l-ol/acetone/ phosphate buffer pH 5 (40/50/I0) Petroleum ether/diethyl ether/acetone (90/10/I) Chloroform/methanol/ Water (65/25/4) Carbon tetrachloride/ Chloroform [95/5) 96% ethanol/water (70/30] Butan. ion exchange etc. pH 5) Silica Gel G 358 Biophysical Chemistry filtration. .1-ol/acet!cacid/ Water (80/20/20) Petroleum ether/Prop-1-ol (99/1) . Two dimensional chromatography may also be carried out much in the same way as descr ibed for . The binder helps in better adhesion of the st ationary phase to the glass or foil plate. Sample application This is absolutely similar to that described for paper chromatography. except th at care should be taken not to scrape the thin layer while applying the sample. however.2} and the methods of elution are much the same as for paper chromatography.2 Certain Solvent System and Adsorbents Used in Thim layer Chromatograph y Compounds Adsorbents Solvent system (/) Mono and disaccharides Triglycerides Phospholipids Cholesterol esters Amino acids Plant pigments Kieselguhr G While preparing stationary phase for adsorption chromatography a binder such as calcium sulphate is mixed with the slurry. Plate Development The choice of solvents (Table 11. The plates are dried after application of the slurry (thin layer for gel £fltration is. The procedure must of course be conducted in a closed cham ber to prevent evaporation of the solvent and the technique used is ascending out of ne cessity. not allowed to dry). the thin layer is activated by heating at 110®C for several hours.Tablell.If adsorption chromatography is to be performed.. with c ertain compounds about 90 minutes may be required. However. Those specific for TLC are: (/) spraying the plate with 25-50% sulph uric acid in ethanol and heating. Many of these have already been named i n the section on paper chromatography (for example. ultraviolet absorption. . The iodine spot disappears rapidly but can be made more permanent by spraying with 0. fluorescen ce. One of the greatest advantages of TLC is the speed at whic h the separation is achieved. These spots Can then be scraped out. This results in charring of most of the compounds. eluted and analysed quantitatively. Generally I0-30 minutes are sufficient.. or production of colors by chemical treatment).5% benzidine solution in absolute ethanol. which sh ow up as brown spots. autoradiography.paper chromatography. if the components are radio-labeled. and (//) iodine vapour is used extensively as a universal reagent f or organic compounds. Detection Several detection methods are available. Iodine vapour is seen concentr ated in the form of a cloud over the region where the components have separated. It may otherwise be used as an aid in order to find out the best conditions for large-scale chromatography. it is often used to follow the course of reactions.Chromatography 359 On plate quantification of the separated components might be achieved by employi ng a densitometer. The ratio of the column diameter to its length also varies. It has also been widely used to resolve plant extracts and many oth er biochemical preparations. which not only measures the ultraviolet or visible absorption of t he separated components but also gives a complete absorption spectrum of the compound for ide ntification purposes. 2. thin layer is more versatile. ion exchange. the apparatus and general techniques used share a lot in common. The com mon features are discussed below. The columns are usually made up of glass or polyacrylate plastic. COLUMN CHROMATOGRAPHY Column chromatography is an often used and routinely carried out technique which is adaptable to all the major types of chromatography. Advutages and Applications Compared to paper chromatography. Thin layer technique has often been used to identify drugs. lengths shorter than 15 cm are also available. It is often used as pilot technique to quickly determine the compl exity of a mixture. exclusion and affinity chromatography are c arried out in a column. The commonly used glass columns have a sintered glass disc at the bottom to supp ort the . faster and more reproducible. radio-chromatogram scanning might be employed to quantit ate the separated components. larger the sample volum e larger the column chosen. Because of its speed and simplicity. Precision made densitometers are now commercially available. Different colu mns differ in their dimensions. Choice of a column of a particular dimension is dictated primarily by the amount of sample. Laboratory columns usually have a diameter of 2-70 mm and a length of 15-150 cm. Although such diverse operat ions as column adsorption. contamina nts and adulterants. Most workers choose a ratio of diameter to length between 1:10 and I: I00. partition. In case t he substance has been radi0-1abeled. On the other hand. which may be si mple or fitted with a ground glass adaptor provides for the eluting solvent to enter the column. T he column is ttted in the upright position and its bottom is sealed with glass wool or such o ther supports. Temperature fluctuations may be harmful for certain chromatographic separations.4). Certain columns are provided with a suitable support under a r eplaceable nylon mesh. For such experiments. The upper part of the column is all the while stirred to ensure . columns with a thermostat jacket are used. of the degassed stationary phase (gel. or resin) is ge ntly poured into the column with its outlet closed. the effluent from the column is led by a capillary tubing to a conti nuous monitor and thence to the collectin8 system (Figure 11. A thick suspension. adsorbent. Packing the Column This constitutes a very critical factor in achieving satisfactory separations. Liquid from a therm ostatically controlled bath set at the required working temperature is circulated through th e jacket continuously for the length of the experiment to maintain the column interior at a constant temperature. called slurry. A cheaper alternative is to use a plug of glass wool with a small am ount of quartz sand and glass beads. The inlet.stationary phase. The column is now filled to about one third its height with the mobile phase. The columns are provided with an inlet and an outlet. This can be achieved by sucking the . This provision decreases the disturbance of the column surface considerably. The slurry is usually added till 3/4 of t he column is full.4 Equipment for column chromatography along with a fractlon collector.360 Biophysical Chemistry Marlotte flask luent Adjustable plunger -Nylon disc Column packing or slntered disc [ : Turntable /.-" Counter-balancing weight Siphon tube (Collection of fixed volumes} --Electrical cable to turntable Figure 11. even packing and to avoid air bubbles. Introduction of Sample It is necessary that the sample to be applied reachs the surface o the column be low the top layer of the solvent. The sample is allo .top layer of the solvent out and then carefully pipetting the sample on to the column surface. The outlet is now opened and the column is stabilized by washing it with mobile phase. Many commercial columns provide an opening at the top. which can be fitted with capillary tubing through which solvent drips into the column. A filter paper disc or nylon gauze is then placed on the surface of the column to prevent disturbance during mobile phase/sample addition. To preve nt the column from drying. a layer of solvent is always maintained above the column surface. so that the height of the solvent in the column can be maintained to a height of 5-I0 cm. Solvent is then added to the column to a height of 5-10 cm. The sample now sinks below the top layer of the solvent to the s urface of the . The column is then connected to a suitable reservoir which contains more solvent.wed to Just run into the column. An alternative a nd better method is to mix the sample with sucrose or flcoll to a concentration of about 1 % to increase sample density. Techniques of Elution: Column Development Continuous passage of a suitable eluant (mobile phase) through the packed column separates the components of the sample applied to the column. Flow-Rate Flow rate. The dye bromophenol blue can be used in place of sucrose.Chromatography 36 ] column. ionic strengt h or polarity of the eluant is changed with respect to time. There are two main techniques of elution: (/) isocratic elut ion. For a satisfactory resoluti on it is absolutely necessary that the eluant flow should be maintained at a stable rate. This process is kn own as column development. two solvents of differin g compositions have to be mixed in correct proportions before entering the column. This is so since with numerous cha nges in the composition of the mobile phase the partition behaviour of the solute molecules is also changing and the cumulative effect of these changes in the partition behaviour i s reflected in the sharper resolution. The easiest way to maintain a stable flow rate is to use a peristaltic pump to force the elu . the process is kn own as isocratic separation. elution with single solvent is many times not suf ficient and a satisfactory resolution may not be obtained. This te chnique leads to a better resolution of the sample components. It is necessary to apply the sample in as less a volume as possible. Alternatively it is possible to reach the column surface directly with the help of a syringe or capillary tub ing. When a single solvent is used as an eluant during development. To produce a suitable gradient. However. and (iO gradient elution. In such cases the pH. An additional precaution is to desalt the sample to avoid anomalous adsorption effe cts. The gradients used could be conti nuous or stepwise. Fe is expressed as the volume of mobile phase per unit time. This is a very important criterion for suitable column development. This process where the composition o f the mobile phase is changed giving rise to a gradient is known as gradient elution. This gives an initial tight band of material when the separation begins and results in a sharper inal separation. This may be achieved by using a commercially available gradient maker. It may therefore be preferable to work with the maximum possible flow rate (indeed this is one of the reasons for the quicker re solution obtained in high performance liquid chromatography as we will see later). However. An increase in the flow rate of the mobile phase.ant on to or out of the column. Analysis and Collecting of Effluent The . color or fluorescence are exp loited in its analysis. vlz. through the column leads to shortening of the time necessary for separation. ultraviolet absorption. commonly used flow rates fall between 30-120 ml/hour cm2. More densely packed columns retar d the flow of the mobile phase and decrease the flow rate. There are two approaches to analysis of solutes in the effluent: The classical a pproach which may be taken recourse to in the absence of a continuous monitoring equipme nt is to collect the effluent in equal fractions and to subsequently analyse each fractio n for the presence and content of the solute. The properties of a particular compound. as it emerges from the column outlet is analysed. However. this approach becomes untenable if the numbe r of fractions . Clearly. It is therefore very important to p ack the column optimally to obtain a good flow rate. Unevenly packed column leads to distor tion of the flow leading to unsatisfactory resolution. Column packing also influences flow rate. The flow rates used differ with respect to the nature of the sample and the nature of the stationary phase. Alternatively th compound may be labeled before application of the sam ple to the column and its radioactivity exploited for its analysis.effluent. an und ue increase in the flow rate decreases the efficiency of resolution and time economy.. To quoittate ech component its peak hekjht at the apex (a) is multiplied with peak width at h alf height (b). Some of these which may be cited as examples are (/) fluorescence det ectors. A wide variety of monitoring equipments each exploiting a different param eter are available. (/v) refractometric detectors. The area under the peak is proportional to the amount of the compound present. Thus as the compound of interest emerg es from the column in the effluent and passes through the flow cell it is depicted on the ch art paper. polarimeters. The electrical signal generated by the detector due to the property of the compo und which it is reading is displayed on a chart recorder. Figure 11. The profde is p by plotting a measurable property of the sample components {absorbance in this diagtum)against the eluted volume as it emerges from the column..5 shows an example of the elution pattern that may be obtained and the way the are a of a peak may be calculated. This area may be determined by measuring the height of the peak and its width at half the height.5 Diogrammatic representation of a typical elution profile. The modem approach. the number of fractions may be in hundreds) or if more than one parameter of the solute is to be studied in each fraction.362 Biophysical Chemistry is too high (many times. and (v) conducti vity detectors. . t herefore. if. etc. the desired sample component is a protein. i. (///) voltameters. is to continuously monitor the effluent coming out of the column. the monitoring equipment may be a UV monitor and may be programmed to read absorptio n at 280nm. For example. one small aliquot diverted to the monitor for analysis while the other stream being collected into differen t fractions for further work. It is common to split the outlet into two streams. The monitoring equip ment is programmed to read the inherent property of the desired compound such as the ult raviolet absorption or radioactivity. Elution volume Figure 11. the volume of effluent collected which corresponds to the apex of the peak. The peaks in such a Ixlttem represent individual zones of sampl e components (proteins in case since the absorbance is measured at 280 nm) separated from each other du ring column developmenL It is possible to identify each component in the pattern on the basis of its elutlon volume.e. In the absence of a method for continuously monitoring the contents of the efflu ent from the column. The contents of these tubes. A range of automaticfract/on collectors are available commercially. If the separation has been satisfactory. can then be pooled together for further study. Collecting effluent fractions manually can be both boring and time consuming. a particular component will be distributed in relatively few number of tubes. They are designed to co llect a definite . which contain the same component. Fractions in small volumes may be collected in different tubes in order to keep the column re solved components separate from each other. it is necessary to analyse it after it is collected in small fractio ns. till the abso rption signal persists. Therefore colu mn efficiency . will signal the fraction collector. will be collected in a single vessei. The whole effluent. Some fraction collectors are fitted with electronic device to measure the number of drops falling in a tu be. If two solute components have very small difference in their partition coefficients they will need a larg e number of equilibrations before they can be completely resolved from each other. a new tube comes into position.ns taki ng place in a column is equal to the number of plates that the column possesses. Other fraction collectors allow the effluent to enter each tube for a interval of time. The fraction collector will now place an empty vessel below the column outlet. Thus. the number of equflibratio. This number can be predetermined so that after a set number of drops have fallen into a give n tube. 363 volume of the effluent in each tube before a new tube is placed in position auto matically. Thus. A better method of fraction collection is by programming the f raction collector for collection of definite volume per tube. The fractions collected ar e subsequently analysed. which is produced by proteins.(B) (c) Chromatography . Ea ch of these segments is called a theoretical plate. Different fraction collectors are programmed to operate in different ways. Thus the column which has a larger number of plates will be better suited for resolving these componen ts whereas a column which has small number of plates will not be as efficient. Concept of Plates: Column Eiciency Let it be assumed that a given column can be divided into a number of identical segments such that one equilibrium distribution takes place on each of these segments. if the desired component to be separat ed is a protein. Recent innovations in fraction collecting systems have led to production of frac tion collectors which are connected to the monitor in a way that the signal for the f raction collector is derived from the monitor signal. and an ultraviolet absorption monitor is being used. the absorption at 280 nm by the effluent. . (C} Increasi ng the number of plates without increasing the column length gives a good resolution with sharp peaks.6). (A) Column wi th small number of plates resolves two substances incompletely. (B) Increasing the number of plates by ncreasing the column length increases the resolution but the peaks are not sharp.is directly related to the number of plates it possesses (Figure 11. I Column lengtl = 15 cm n = 80 Plates Column length = 30 cm n = 160 Plates Column length 15 cm 160 Plates Elution volume Column length and number of plates as related to c)lumn e.jciency. This may be done by making a better column and optimizing other factors which affect the solute interaction with the two phases. there is also considerable band broadening (Figure 11. temperature. A short list is provided in Table 11. the the solute between the two phases number of plates that a column has How can one increase the number of plates to increase the efficiency of a column ? One way is to. TYPES OF CHROMATOGRAPHY 1.. No electrostatic forces are used by the adsorbent to attract molecule s to its surface. ADSORPTION CHROMATOGRAPHY A solid.3. vtz. which has the property of holding molecules at its surface.. we have already seen that the nature temperature and many other factors influence equilibration of and therefore these factors will also have a bearing on the . method of sample introduction etc. . This optimization of column development condi tions would lead to increased number of equilibrations taking place thereby increasing the n umber of theoretical plates in the column. However. increase the number of plates without increasing the length of the column. Adsorbents A range of materials is available but a few common ones are sufficient for most purposes. however. One is the different degree of adsorption of various components on the adsorbent surface and the other is the varying solubility of different components in the solvent used (mobile phase}.364 Biophysical Chemistry It would seem that the number of plates would uilt. depend only on how the column is b of the solute. Although this way one might get a better resolution. This is so since the number of plates is directly proportional to the column length.lengthen the column. Adsorption can be fairly specific so that one solute may be adsorbed selectively from a mixture.6 C). the flow rate. We can. This approach of increasing the number of plat es is therefore obviously a better approach because the band broadening due to longer columns wi ll be avoided and one may be able to get resolution in narrow peaks (Figure 11. can be desc ribed as an adsorbent. Two differing factors are exploited in separation of components by adsorption ch romatography. flo w rate.6 B). the ad sorbent has to be activated by heating it for several hours at 110°C. The adsorptive activity of an adsorbent can actually be controlled by varying the amount of water it contains. Certain adsorbents might not be inert but react and degrade components to be separated during experiment. however. In addition to relative solubilities of the solute i n the eluting solvent.Table 11. 360°CI for about 4-5 hours and then allowing the dehydrated mate rial to pick up a suitable amount of water. Certain other ad sorbents might imbibe water from the atmosphere and become useless. the sample should be introduced in a solvent in which it is highly soluble. which might be detrimental for separation . Solvents Virtually any organic solvent can be used as the mobile phase. it is necessary to consider the competition between the solutes and the solvent for the . As far as possibl e. and as such care must be exercised while selecting one. presence of water results in better separation since adsorption is coupled with partition du e to the presence of water. Sometimes. In such cases. this hel ps to keep the sample volume at a minimum.g. Thus we can prepare a tailored adsorbent by dehydra ting it at high temperature (e.3 Some Common Adsorbents in Order of Increasing Adsorbing Power Powdered cellulose Starch Sucrose Calcium Carbonate Magnesia Silica Gel Alumina Adsorbents can possess some qualities. From a column chromatography point ofvlew. However they also decrease the resolving power of the colunm since they do not close together. be carried out using column. then. fortunate that a long list of solvents is available (Table 11. Tble 11. ' Adsorbents for thin layer chromatography are often impregnated with various long during . while the solvent eluting the solutes very slowly will lead to uncomfortably long retention times which will result into excessive band broaden ing and sample dilution.Ethyl Acetate Acetone Ethanol Methanol Water Organic acids & bases Adsorption chromatography can.4). Traditionally used adsorbents are composed of irregular shaped.4 Some Common Solvents in Order of Inreasing Eluting Power fom Alumina Petroleum Ether Carbon Tetrachloride Tri-Chloro Ethylene Toluene Benzene Chloroform Ether . It is. It is dear. a solvent which e lutes the solutes too fast will give a poor separation. TLC. or paper techniques.Chromatography 365 adsorption sites on the surface of the stationary phase. these particles allow adequate flow. Thus. that the choice of the solvent has to be a compromis e between numerous mutually opposing factors affecting chromatographic separation. therefore . A balance has to be reached between a cl#ser pacing and adequate eluant successful separation through adsorption chromatography. techniques such as ion-exchange chromatography. mono-. a nd adsorption chromatography because of their greater However. tig htly held on which migrated the mobile phase which was another solvent of lesser polarity. The list includes amino acids. true for column chromatography when silica gel or powdered cellulose is the soli d separation achieved in liquid-liquid chromatography is therefore based upon the the immiscible solvent systems. Thus. neutral lipids. recently. ?aper chromatography. the fact that it is highly empirical and non-reproducible because of variations in the of adsorbents. . cholesterol easters. the stationar y phase particles. The stationary phase there was a polar solvent. Further discussion chromatography is avoided here for fear of repetition since most of the aspects been covered under paper chromatography. The disadvantage with adsorption chromatography. phospholipids. when thin layer chromatography is performed by silica gel layer. adsorption chromatography has re-emerged as a powerful separation times in the form of high precision liquid chromatography (HPLC. Adsorption chromatography has been extensively used for biochemical separations. PARTITION CHROMATOGRAPHY Liquid-Liquid Chromatography We have already covered one form of liquid-liquid chromatography in detail previ ously. Papers coated with different adsorbents are commercially available and the techn ique has already been described previously. This too then is a form of liquid-liquid chromatography. This enhances the separation achieved. -J 2. however. . water. see later} sometimes applies adsorption principle for sample resolution. etc. and disaccharides.)reparation. may be noted. The solid support w hich is coated on to the inside surface of a long column is inert to the sample components and doe s not react with them in any way.7 Carrier 366 Biophysical ChemistryI One point.To detector Gas (C) Column Figure 11.! Ir such situations it is advantageous to use non-polar organic solvent as the st ationary phase and aqueous solvent as the mobile phase. it merely acts to hold the liquid phase in a stable disper sed form so as to present a large contact area. phase is a non-volatile liquid. In other words. the nature of the phase s is reversed. Gas-Liquid Chromatography (GLC) The basis for the separation of the compounds in gas-liquid chromatography is th e difference in the partition coefficients of volatilized compounds in the liquid stationary phase.i' Gas-liquid chromatography is a form of column chromatography where the stationar y. In such cases separation wi ll not be achieved.i The technique is aptly known as reverse-phase chromatography. A . B. The non-polar soli d support for such chromatography is usually powdered rubber coated with benzene. If during the separation of predominan tly non-polar solutes through partition chromatography. which is aqueous. The mobi le phase. however. Thi phase is dispersed over a surface of an inert so//d support. It is the liquid phase that interacts with sample components. The stationary phase here is known as the liquid phase. the stationary phase is polar. the sol utes will not! have much solubility in it and will therefore migrate fast with the mobile phase in which they! are more soluble (since it is relatively non-polar). flows over this stationary phase. To detector {A} Column (B) " To detector Solid Liquid support phase Diagrammatic representation of gas chromatographic separation of sample componen ts on the basis of their partltkm coefficients.':: 'v":: . the three components migrate on the column. the gas. (B) As the gas continues to flow over the liquid phase. Vertlcl position of the arrows indicates the distribution (partition) coefficient of the compnents. :" ' ::". When a small quantity of the volatile sample is introduced int. In the Column Gas "'. The component indicated by the second arrow interacts equally with both phases. The situation is ex'tly reverse for the components indicated by the third ar row. the gas promptly carries it on to the column (hence the name "carr/er g as"}.gas stream termed as the carrier gas flows continuously through this column at a flow rate which is controlled (Figure 11.7). . the component by theflrst arrow interacts more with the liquid phase and less with the gas pha se. (C) The components have become widely separated from one another.:. (A) Sample consisting of three components is introd uced in the column. which is coated on t o . The magnitude of the detector signal as measured by a continu ous recorder when plotted against the time taken by .. The char acteristic sition of the peak is measured in terms of the volume of gas that has traversed through the column between the time that the sample was applied and the time at which the pa rticular !omponent emerged from the column. (B). A chromatogr am is made up of a number of peaks. which is present in the GLC apparatus. the area of the p eak is proportional to the concentration of the component causing it. each of which is due to a component of the sample. the sample components become distributed between the liquid and the gas phase. There is a variation of gas liquid chromatography which is knon as gas-solid . Here. This gas volume has been given the term reten tion volume.w hich reads the concentration of a given component present in the carrier gas and converts i t to an quivalent electrical signal. A typkxzl gas chromatogram. The solid phase.8 i Chromatography 367 icolumn. As said for column chromatography previously. (C). These separated components eventually elute out of the column and reach the detector. the component distributing more in the liquid phase is ret arded more and the component prefering the gas phase is retarded less. and (E) are detector skjnals due to different sample components which emerge from the column at different times depending on their pa rtition coefficients. ef fect is different for different components. (D). These components therefore travel more slowly than the carrier gas because they are being retarded by virtue of their interaction with the liquid phase. fundamental to gas chromatography and may be used for the ana lysis of a ple (see later).the particular component to elute out of the column produces a pattern which may be called a chromatogram (Figure 118). (GSC). !V. The position each of the peak is characteristic of the component to which it is due. In the long column. the liquid phase is absent.I 1. (A). these sample componen ts ecome separated from each other on the basis of differences in the retarding eff ect. The retarding. This term is. This eventually leads to their separation other.is not inert in this technique. . The sample components therefore become between the gas and the solid surface on the basis of differences in the the solid surface. it interactswith the sample components by the gas by exerting adsorption forces. (F) FractWn collector. Gas used at high density gives a better separation but takes a long time to achieve it. Even steam is used for special purposes. (C} Sample injection chamber. A soap film flow meter is used almost exclusively to measure the flow rate. helium. The usual contaminant is water vapour. The sample components separate in the column as discussed above and pass through the detector which gives signal to the recorder. injector. The gas is usually passed through the column at a rate of 40-80 cmS/mmute.368 The essential components of a gas chromatograph are shown in Figure 11. The final passage of the gas is through th e flow meter (not shown in the Figure 11. . the carrier gas constitutes the mobile phase and provides transportation for the sample components through the apparatus.9. Since the procedure is usually ca rried out at high temperatures. The individual solute components can be coll ected in a fraction collector for further study. The in ert carrier gas stored in a gas tank passes through the pressure regulator into the sample injection chamber from where it carries the sample onto the column. Most commonly used gasses are nitrogen and argon.9 Schematic of a gas chromatograph. The column is usually packed with highly porous solid onto which is coated a thin film of liquid used as the stati onary phase. (A) Carrier gas tank. (D) Column. Figure 11. A low gas might give a faster separation which will not be as satisfactory. and (G) Reco rder Carrier Gas In GLC. a thermostatted oven is provided for the column. Passage through a cold molecula r sieve trap is very effective for removing last traces of water vapour.9) into the atmosphere. but. (E) Detector. t he choice gas usually depends on the requirements of the detector and also on the availabi lity of the gas. an d the detector. The gas must be chemically inert and pu re. Purity of the carrier gas is critical since even a small impurity can give rise to noise in the detector. hydrogen and carb on dioxide are also used. However. (B) Pressure regulator. 3.4. copper or glass tubing. 1.6.Columns Two distinct types of columns are commonly used.2.5 mm bore. The op en tubular colunms are also known as cap///anj columns. 6. or 9. and anywhere between 1-15 meters in . Packed colunms are stainles s steel. packed and open tubu/ar. the unrest ricted gas flow is retained. These columns have limited sample capacity and are unsuitable for large-scale separati ons. i. In these columns a porous layer is formed on the inside wall of the tubing. The inner wall of these columns is coated with the liquid stationary p hase to about 1 tm in thickness. The inert porous layer is then impregnated with the liquid stationa ry phase. bltd Support An ideal support should be chemically inert although wettable by the liquid phas e so that it will spread in a thin layer of uniform thickness.. The supp ort is coated in such a way that the inherent property of the capillary columns. The second type is known as support doated open tubular columns (SCOT). Open tubular or capillary columns have open unrestricted path for the gas within the column. In addition it must be thermally stable and mechanically strong.+ (MeSi)2NI-I ---> Me--SI--Me Me--SI--Me O Chromatography 369 length. These columns are about 15-30 meters in length with an inside diameter o f about 0. It should ideally have a hi gh specific surface {I m2/g). One is known as th e wal/coated open tubu/ar co/umn (WCOT) in which the liquid phase is coated on the column wai l.25 ram. Columns are bent in U or W shape or coted to fit in the oven.OH e Me ---SI---O--Si-. The open tubular columns are of two kinds. The geometric configuration of the column is of little concern as long as the be nds are not sharp. They are filled with narrowly sieved inert support coated with a liquid phase fo r GLC. They . The porous layer can ei ther be formed by chemical treatment of the inner wall or is deposited on the inner wall. For preparative purposes packed cohmms of several inches diameter might be used.e. These columns have a higher sample capacity. The most commonly used supports are derived from diatomaceous earth and teflon. 5 lists properties of s ome principal types of solid supports. The sfla nol (Si---OH) groups that cover the surface of the diatomaceous earths are very polar and tend to react with polar solutes. Ac id washing is effective in removing mineral impurities which might otherwise serve as adsorption sites. Embacel etc. Some other solid supports are known by the trade names Celite 545. such as Chromosorb P (a form of crushed commercial firebrick) and Gas Chrom R. The diatomaceou s earth supports may be either Firebrick derived materials. Deactivation of all diatomaceous supports is necessary for most applications. ff the sample is polar further treatment of the support might be required. Treatment with hexmethyldisflazane (HMDS) sialinizes the surface and makes the surface inert to polar solutes. and Gas Chrom Q.. (all diatomaceous earth derived). Anakrom ABS. This treatment is all that is needed if the sample to be separated is non -polar. Table 11. wh ich include Chromosorb W (diatomaceous earth heated with an alkaline flux). Phasep P. However. or materials derived from filter aids. .are sold under many trade names depending on their pretreatment. 500 2-11 3O 1 20 . paraffin) Steroid. alkaloids.W. ethers.Silicon rubber gum (SE-30) Aplezon L (High M.05 1 . high boiling hydrocarbons Amines. cyclic hydrocarbons 370 Biophysical Chemistry TabIe 11. alcohols All types All types Hydrocarbons ' Hydrocarbons. ketones.01-0. fluorides Oleflns.5 Some Properties of Principal Types of Solid Supports Used in GLC Type of solid Support Surface area Approximate maximum liquid phase loading Percent by weight Diatomaceous earths Crushed firebrick Glass micro spheres Porous silica beads (PORASIL] Polytetrafluoro ethylene 1-3 4-6 0. pesticides Esters. Very often powdered teflo n is used as a support for very polar solutes. Obviously one single liquid cannot meet all the requirements stated above. This is so since the gas phase is inert. (i//) it should provide ap propriate partition coefficient values for the components.6 Common Stationary (Liquid) Phases for GLC Stationary phase Typical ssmples Max. Thus. Table 11.6. °C 350 Carbowax 20 M (polyethylene glycol} Silicone off (DC-550) Dmonyl phthalate Sqalane . Very lightly loaded gl ass beads are preferred when very rapid separations are desired. A few liquids commonly used are listed in Table 11. to select the liquid phase it is necessary to match the polarity of the stationary phase a nd the sample components of intercst.2O Special supports are popular for particular applications. others for high temperatures.0f interest. Graphitized carbon and carborundum are other sup ports for special applications. the separation occurs only in the liquid phase. The requirements for a good liquid phase are: (0 it must be non-volatile at the temperature it is to be used. Some are needed for low temperature. Temp. and (/v) it should be complet ely inert towards the solutes. (it) it should be thermally stable. Liqu/d Phase A good rule to follow when selecting liquid phases is "like dissolves like". A g ood separation will occur only when the sample dissolves well in the liquid stationa ry phase. Hexadecane Silver nitrate in propylene glycol 300 250 200 150 140 Coating the Support To prepare the column packing. correct amount of liquid. acetone) is added to the solid support. The mixture is then he ated very slowly with continuous stirring in order to evaporate the solvent.phase dissolved in a low boiling solvent (pentane. the last traces of s olvent are removed . Since this part o the apparatus is under pressure. The open tubular column has a much lesser capacity and utilizes very small samples of the order of 10-3 to 10-2 ml. Open tubular columns contain no packing. If the sample is not alr eady a vapour. COOH. However. This results in a thin layer of the liq uid phase. enough heat must be supplied to establish vaporization immediately. heptane. continuous motion of t he plunger is the byword. due Care must be exercised while operating the plunger. Liquid samples dissolved in a suitable organic sol vent such as ether. The column is gently shaken and tapped all the while to ensure an even packing. Sometimes pressure up to 5 p. Solid samples are much more difficult to introduce. To apply a thin coat.Chromatography 371 under vacuum. Sample should ideally be introduced in the vapour form. amino acids and fatty acids and increase the volatility and distr ibution coefficients of these compounds. The best method is to seal t hem into a thin vial which can then be introduced into the injection port and then crushe d from outside.s.column is bent or coiled to an appropriate shape that will fit in the oven. The above named methods are very useful for carbohydrates. Both the ends are plugged with glass wool and the . Excess solution may t hen be evaporated by passing a hot carrier gas.L may also be applied. Columns are filled by pouring the packing into straightened column . Sample Preparation and Introduction If the sampIes are non-polar or have a very low polarity they may not need any pretreatment. --NH2. pretreatment is needed (in the absence of pretreatment these sample components would be retained on the column for excessive time which will result in poor sep aration and peak "ta///ng").. An average packed column of 9. . a dilute solution is forced through the column at a very slow rate. These fuIctional groups are subject to derivatization methods s uch a metylation. A swift. trifluoromethylsflanization etc. and methanol are injected from a syringe through a rubber septum into a small heated chamber immediately preceding the column.4 mm bore coupled to a thermal conductivity detector optimally utilizesa sample of 1-50 ml or less. sflanization. Size of the sample varies depending upon the column and the detector used. if the sample possesses such polar functional groups as -OH. etc. of liquid phase. The principle of flame ionization dete ctor is illustrated in Figure 11. the jet of which forms one electrode. The detector output is then suit ably amplified and is traced on a strip chart recorder. The temperature of the thermostatted detector should be maintained at a le vel high enough to prevent condensation of high boiling vapours. This is by far the most widely used detector. (i) ionization detector. It measur es all organic compounds and it can detect as low as one nanogram of any given c ompound. This results in chromatogram of concent ration versus time. At the same time it shou ld not be so high as to cause decomposition of eluent material. the asso ciated electronics are complicated and moderately expensive. Three most commonly used dete ctors are described below. either used as carrier or introduced into the detector through elsewhere. As the resistance of the flame is very high and the current generated is weak. the detector detects the presence of the individual components as they leave the column. The sample com ponents becomes ionized in the flame and give rise to a cUrrent between the two electrod es.Detectors Located at the exit of the separation column. The other electrod e is mounted just near the tiP of the flame and consists of a platinum wire. . The flame changes col or the moment a separated component comes out of the column and into the flame. is bur nt to give a nearly colorless flame. Hydrogen.10. The electrons produced give rise to a curr ent across the electrodes to which a suitable voltage is applied. whic h has the ability to capture electrons. Obviously only those substances having an electrons capture capability can be measured by this type of detector. dieldrin.25 cm above the plasma Jet. This detector has radioactive source (3Ni} which ionizes the carrier gas coming out of the column. This low temperature source suppresses the normal flame ionization response of the co mpounds not containing nltrogen or phosphorous. This bead is electrically heated to about 600-800 °C . This design allows adjustment of the bead temperature independent of the plasma as a source of thermal energy. it captures the ionized e lectrons thereby causing a drop in the current. Radioactive source Fure 1 I. It is mostly used to measure polyhalogenated compounds.372 Gas flow Biophysical Chemistry lgure 11. the detector responds to both nitr ogen and phosphorous containing compounds by greatly enhancing the ionic dissociation of rubidium . The detector design is illustrated in Figure 1 I. 11 Desk3n of electron capture detector (///) Therm/on/C errgss/on detector. 1 I. and aldrine. A non-volatile rubidium silicate bead is centered about 1. When a sample component. It is very sensitive and can detect as littl e as one picogram of these compounds. comes out of the column.10 Flame onrtion detector (li) Electron capture detector. particularly p esticides such as DDT. With vanishingly small hydrogen flow. This detector employs fuel-poor hydrogen pl asma. This change in the current is measured and record ed. Chromatography 373 chloride. the d etector may be replaced by a mass spectrometer. There is thus a linear relationship between the logarithm of the retention time and the number of carbon atoms which can be exploited to some extent to the identification of u nknown compounds. Consequently it is used to measure organophosphorous pesticides. retention time aids in qualitative analysis in gas chromatograp hy. The time of emergence of the sample relative to that of the standard is determined to give t he relative retention time ( t. lure 11. However. from the point of view of a positive identification. Rf (see section on paper chromatography). Unlike paper chromatography. this relationship is limited to members of a ho mologous series.and Qualitative Analsis Much in the same manner as the retardation factor.12 Dependence of resolutn f two peaks n sepamtn factr and number f plate s in clumn (A Pr . One can therefore identify unknown compounds by comparing their tR with that of the known compound s.. Under standard conditions of temperature. if it were possible to relate the retention characteristics to the structural properties of the molecule. In cases where the identity of the compounds being separated is not known. it is difficult to reproduce conditions exactly from one r un to another in GLC. for example the methyl esters of the saturated fatty acids. the time taken for a compound to emerge from a column is constant and iS-known as the retention time. gas flow. gas compressibility etc. The simplest and the most reliable relationship of this kind is the one which depend s upon the number of carbon atoms. Special separators remove the bulk of th e carrier gas from the sample emerging from the column prior to its entry into the mass spectr ometer. This difficulty is tackled by analysing a standardcompound with the sample. Less than one picogram of compounds containing nitrogen or phosphorous can be detected. Each additio nal CH2 increment is known to decrease the vapour pressure of the components by a consta nt amount. It would be more useful. The value of tis constant for a given column under different conditions. Retention Time . separatl factor. . (B) Good separation factor. large number of p/ares. (D) Good separatWnfactor. large number of plates. (C) Poor sepamtlonfactor. small number of plates. small numbe r of plates. In addition. However. m s which make it possible to collect appreciable amounts of individual components of samp le mixtures. the resolution can be improved by increasing the number of t heoretical plates. Inpractice. a splitting system is established at the end of the column. GSC Versus GLC The problems and advantages of GSC visa vis GLC are outlined below. Repeated chromatography of the sample mixture is carried out for this purpose. The two techniques are more or less similar to each other. the instrument ation for GSC is identical to GLC with very minor differences. the primary differenc e being that the partition within the column is caused by adsorption on a solid surface rather th an solubility in a liquid phase. variations in stationary phase and the number of plate s is used for a successful resolution (see Figure I 1.12). Consequently the retention times are inconveniently long in GSC. retention times are considerably smaller when working with such gases a . F or preparative GLC. Gas-Solid Chromatography (GSC) Gas-solid chromatography actually preceded gas-liquid chromatography by several years. This splitting system splits the gas stream emerging from the column in such a manner that a small part of the st ream goes into the detector (to be monitored) and the rest goes to the collector. This ratio is known as separation factor. The coll ector possesses a liquid nitrogen trap (to liquify the vaporized components by cooling) in which t he individual components are collected. The separation factor is a func tion of the stationary phase and can be varied in order to improve resolution by varying the stationary phase. Quantity of a sample component might be determined by analysis of its peak area.374 Biophysical Chemistry The separation of two components of a sample is a function of the ratio of their retention times. both. (/) Distribution coefficients of components in GSC are much larger than the partition coefficients of GLC. Since the techniques are similar in most aspects. Preparative GLC GLC can be made preparative by usage of suitable columns and collection syste. Thls often leads to pyrolyzation of samp les in GSC. esolution is poor. solvents. nitrogen.. volatile vegetable otis and organic ac ids etc. (v) GSC suffers from lack of reproducibility. argon. In other cases the sample is irreversibly adsorbed and no resolution can ta ke place.s hydrogen. for which it is routinely used. and oxygen {these gases give too short a retention ti me in (i The upper temperature limit in GLC is limited by the necessity of retain ing the liquid stationary phase. atmospheric pollutants . plant extracts. since there is no liquid phase in GSC. Small concentrations of samples are. ('t) GSC should better not be carried out with high concentrations of samples as this gives skewed peaks and the r. considerably h igher temperatures may be used. However. The retention behaviour consequently differs greatly leading to approximate theoretical interpretation. gas chromatography is being increasingly used as an analytica l tool to study . resolved well. The size and shape of the area of solid surface and also the composit ion of the surface layer varies greatly from one experiment to the other. Application of Gas Chromatography Apart from the separation of components of tobacco smoke. The upper limit in this case is limited only by therma l instability of the sample or materials constituting the apparatus itself. (iv) Specific areas of solid adsorbents may be as much as 100 m2/g (about 100 times in excess of solid supports used for GLC). however. essential oils. as solid surfaces are very dif ficult to reproduce. . (/0 Gas chromatography is one of the most widely used procedures to study re action rates. (/v) Gas chromatography is today being widely used to analyse such molecular properties as (a) vapour pressure. The sepa rated and labeled compounds can then be collected from the column exit. Kinetic data in these cases become available directly from elution behaviour. however. Thus gas chromatography is serving as an excellent tool to study thermodynamic properties of solutions. applications have been carrie d out on substances of non-biological interest. the ongoing developments in the field of gas chromatography will allow the same applications to be performed on areas of biological interest also. aCl). However. liquid crystals.Spectrometry (GC-MS} The use of retention data for identification is useful in those cases where the chemical type of the sample is known and when there are a number of pure compounds of this type avail able for the . and gas-liquid i nterracil adsorption have found GLC to be a tool of importance. (e)bond angle deformation.Chromatography 3 75 many parameters. energies and mechanisms. use a GC column in which mixture of unlabeled compo unds is injected. (d) molecular geometry. the preparation of small quantities of several such species by conventional techniqu es is difficult.g. (/) An ever expanding number of workers in the fields of solution chemistry including study of polymers. and 0 ionization pdtential and el ectron Gas Chromatography . There are instances in which a solute has been made to react or interact on the GC column in a rate controlled manner. The GC column contains the exchanging nucleus (e. It needs to be pointed out that although most of such analytical. (c) molecular weigh t. One can. util ity. Lewis acid-base properties. Isotope la beled organic and inorganic compounds are used in many biological fields. ('gO GC columns can be made to behave as isotope exchange vessels. A short list of such analytical applications is provided below. (b) heat of vaporization. if not indispensable. There are a number of possible arrangements. ff the comparison method fails in iden tification of the unknown s. a high resolution magnetic sector field mass spectrometer is needed. the gas stream exiting from the coiumn is passed to th e spectrometer ion source via an intermediate stage that reduces the carrier gas pressure to the high vacu um conditions required by . which remove a large fraction of carrier gas by diffusion. The molecular separators are usually of three kinds (0 effusion separator. in complex mixtur es there are several possible interpretations for each peak and so the agreement between the retentio n characteristics of a known standard and any component can never be regarded as conclusive evidence of identification. and (//) they remove the gas molecules. The intermediate stage might involve the use of molecul ar separators. The separators achieve two ends. (/) they remove the gas from the sample components thereby enriching the component. and quadrupole mass filters. use of an ancillary technique for identification of compo nents is required.mass spectrometry (GC-MS) uni ts have become the norm of the day. There seems to be no clear preferenc e for one or the other type of instrument. To remove this ambiguity.pectrum. However. The molecular peak and the fragment peaks form a typical pattern that can either beinterpreted by experienc e or by comparison of the unknown spectrum in the library. However. This t ype has become so popular that directly coupled gas chromatography . In general. which might interfere in sample identifi cation. For GC/MS usually low resolution spectra are sufficient. The ancillary technique most uded in modern instruments is mass spectrometry. more often than not. depending on the type of column use d and the mass spectrometer. magnetic sector mass spectrometers. which reaches the ma ss spectrometer. . the mass spectrometer. Two types of mass spectrometers are mainly used for GC/MS work.determination of reference date. (/0 Jet/orifice sep arator. and (t/0 membrane separator. If the mixture of molecules of different size is plac ed on the top of such an equilibrated column. gel filtration. The method is also known as molecular sieve. The many advantages that this method has over other separation p rocedures have resulted in its widespread application. or molecular exclus ion chromatography.Molecule too large to enter the pore Intermediate size molecules i are partially excluded Path of a Path of a large molecule. the larger molecules pass through the interstitial spaces between Figure I I. (ii) almost 100% solute rec overy (iii) excellent reproducibility. The advantages are: (/) gentleness of the technique permitting separation of a labile molecular species.lusion of large molecule. 13 Illustration of prlnclple of moleculox sieving. and (iv) comparatively short time and relativel y inexpensive equipment needed for its opcration It is a powerful separation procedure so that samples differing by about 25% in molecular dimensions may be totally separated by this technique on a single gel bed. . Principle Gel permeation chromatography is based on a very simple principle. GEL PERbIFTION CHRObiTOGIPHY Gel permeation chromatography is a separation method dependent upon molecular si ze. elutes fast (B} elutes very sloWly 376 Biophysical Chemistry 1. A column of g el beads or porous glass granules is allowed to attain equilibrium with a solvent s uitable' forthe molecules to be separated. (A) Schematic rep resentation of exc. small molecule. .(B) Effect of partlcle size on their elution rates. a s said above. the technical term for this is vo/d volume (Figure I I. This point can be better understood when we consider the fact tha t linear polysaccharides and ibrous proteins have a lower exclusion limit in a given gel as compared to globular proteins of comparable molecular weight. This is because the pores of the gel have smaller diameter than what is needed for the large molecules to enter. This molecular dimension dependent variation ofKbetween 0 and I makes i t possible to separate solutes within a narrow molecular size range on a given gel. do not enter th e gel and are said to be excluded. is known as V (Figure 11. The distribution coefficient. One can therefore describe the exclusion limit of a gel as the molecular weight of the smallest molecule incapable of entering the gel pores. however. also see Box 11. however. therefore. is . The degree of retardation of a molecule is proportional to the ti me it spends inside the gel pores. It. (Figure 11.Chromatography 377 the beads. It is usual to judge the exclusion limit by molecular weights (although Stokes' radii are more accurate) because su ch data is easily available. Large molecules. 14). D ue to variations in pore size for a given gel. The lower limit for effective use of molecular sieve gels is usually about 10% of their exclusion limits. Kd = 0 w hen the solute molecule is large and completely excluded from the inner solvent../. the inner solvent. move down the column w ith little resistance. For a given type of gel. the solvent surrounding the gel beads is indic ated as Vo. does not mean that molecular weights are a true r eflection of Stokes" radii. Kd. Le. which is a function of the molecule's size and the pore di ameter.e. The K value in such cases wil l vary between 0 and 1. K = I if the s olute molecule Is small enough to penetrate the gel pores and diffuse into the inner solvent. can enter the pores and are thereby ef fectively removed from the stream of the elutlng solvent. there is some inner solvent that will be available and some that will not be available to solutes of intermediate sizes. which is a function of its molecular size. The small molecules. These molecules are thus retarded. the distribution of a s olute particle between the inner and outer solvent (solvent within and outside the gel bead) is defined by a dstrlbution coefficient.14).. The volume of outer solvent. A few simple and useful mathematical relationships about solute behaviour on mol ecular sieve gels are described below.13.3). The molecules with Stokes' rad//equal to or exceeding pore diameter. The volume of solve nt inside gel. 11.14 Diagramatic. is dependent upon these three variable s. Fig.symbolized by Kd. respres . The effluent volume. Thus. .nuion of solvent insle the gel bead (V). vokl v olume (Vo} and total volume VJ. V. a = the dry weight of the gel. therefore. is given by v. (/0 It should preferably contain vanishingly small number of ionic groups. Sagavac). the sample volume applied for complete separation of two substances should not exceed Vs. Thus where. Blophystco Chemistry The volume of inner solvent. The distribution coefficient and the effluent volume are both related to molecular weight of the molecules being separated. differ ent distribution coefficients (Kd and Kd2) the difference in their effluent volumes.. However.therefore. (/v) A given gel should have uniform particle and pore sizes.KJv. Types of Gels A gel filtration medium should possess the following characteristics: (/) The gel material should be chemically inert. . there are five principal types of media that fulfill the criteria to quite some extent. (v) The gel matrix should have high mechanical rigidity. The value ofKd is characteristic of a given molecule and does not vary with the geometry of the gel bed. the numerical value of V is dependent upon the size of the column. They include (0 cross-linked dextrans (trade name Sephadex).. (it) agarose (Sepharose. Vcan be calculated if the dry weight of gel employe d and the water rega/n va/ue of the particular gel are known. Wr = water regain value. Vs. Bio-Gel A. = (K. For two substances possessing different molecular weights and. Thus. One can therefore calculate molecular w eight of molecules if one determines the effluent volume by gel filtration experiment (se e applications of gel permeation chromatography). 378. No such material is available which will fulfill all the above criteria satisfac torily. However. . (///) Gel material should provide a wide choice of pore and particle sizes. include macroreticular polyvinyl acetate (Merck-O-Gel OR). (iv). For proteins and most of the bio-molecules. Each of the glucose residues in the polymer possesses three hydroxyl groups givi ng the dextran . Other gels. and (v) polystyrene (Styragel. porous glass and silica granules Bio-Glas Porasil). Sephadx. mircroparticulate alu minas and silica's (Spherisorb). which have been used for gel filtration.form (bead celluloses hav e not been developed -specially for gel filtration but are known to have molecular sieving properties }. When Leuconostoc mesenteroids indulge in sucrose fermentation. are used to prepar e Sephadex. These polymers. Sephadex is by far the most popular of all the gels. known as dextrans.(rio polyacrylamlde (Bio-Gel P). and eellullosepackings in bead . BioBeads S). large polymers of glucose are the result. the amount of water taken up in the completely swollen gel granules by one gram of Sephadex} of the gel multiplied by a factor of I0 (Table 11. various classes of gel beads with exclusion limits between . Table 11. and formamide.15 Structure of cross-linked polydextran (sephadex) a polar character (Figure 11. Sephadex gels will swell in glycol. d/methylsulfox ide.CHOH OH l o °r o OH OH Fjure 11.15)..0 . In addition to water in which they are normally used. * G-25 G-50 G-75 G-100 G-200 2.7 13/fferent Types of Sephadex Gels Eclus/on llm/t Mol.7). CHCHCHCI.1 and 200.10. These gels are identified by a number such as G. By controlling the cross linking reaction .000 daltons can be produce d. or G-200. which refersto the water reg ain value (Le. and are stable in bases. Wt. %/ Pore size is controlled by the molecular weight of the dextran and the amount of epichlorhydrinused in the preparation.5 5. Sephadex gels are insolubl e in water. weak acids and mild reducing and oxidizing agents. The agent used for cross-linking dextran polym ers is epichlorhydrin. 0 20.000 5.7.I0.000 I00 5.50. .000 I0.0 5. Inc.000 500.I00.000 .000 *Manufactured By Pharmacia.000 50.000 1.000 I00.000.000 5.000 200.000 -200.5 I0. the large pored gels used for such separations lack in mechanical rigid ity. However.0 00 daltons. Polyacrylamide is insoluble in water and common organic sol vents and may be used in the pH range of 2-1 I.8 Commonly Used Polyacrylamide Gels Water regain g/g dry gel} Exclusion lim/t Mol. This very popular medium is produced by polymerizing acrylamide into bead form. These gels. Table I 1. Polyacrylamide gels are usually identified by a number such as P-10 or P-100 which if multiplied by a factor of 1000 will indicate the exclusion limit of the gel in thousands of daltons.1 .Proteins 380 Biophysical Chemistry Polyacrylamide. Polyacrylamide gels can be used to separate molecules of up to 300.100 Bio-Gel P-200 Bio-Gel P-300 5. Wt. Limits *Bio-Gel P. are unstable to bases due to hydrolysis of amide groups. Wt. This detrimental property ma y cause very low flow rates. however. Fraction range Mol. A brief discussion of the control of the bead and pore size of these gels is provided in chapter 12.I0 Bio-Gel P-60 Bio-Gel P. Table 11.8 lists the commonly used polyacrylamide gels. These beads also tend to become compressed in the column. In fact reports are available whe re these gels have been used even at pH 13. They are therefore almost completely inert to the solutes being separated.6.000 .lo0. by virtue of their greater porosity. and owe t heir gelling properties to hydrogen bonding of both inter-and intra-molecular type.9 lists c ommonly used agarose gels.O00 300. however. Thus.000 daltons.5 22.000 30.5 13.000 . wide use has been made of these gels in the study of viru ses.9 Commonlv Used Agarose Gels .0O0 . has to be taken about controlling temperature when using agarose.000 200.000 . and polysaccharides.400. They are linear polysaccharides of alternating residues of D-galactose and 3. The chromatography has therefore to be performed between 0° and 30°C.000 100.30O.000 *Manufactured by Bio-Rad Laboratories Agarose. agarose gels.0 I0.O00 80. Agarose gels areproduced from agar.6-anhydro-L-galactose units.000 . In contrast to Sephadex.17. which cannot be used to separate biopolymers larger than 300. Table 11. Care. Freezing temperatures and temperatures higher than 30°C cause alterations in the gel struct ure.000 60. nucleic acids. Table 11.O00 40. These gel s are hydrophilic and are almost completely free of charged groups.00O IO0. may be used to separate molecules and particles up to a molecular weig ht of several million daltons.8 7.000 5. High salt concentrations do not usually affect the se gels adversely. The-agarose gels are compatible with all aqueous buf fers and are completely stable within the pH range of 4-10.70. wt.} Polysaccharides *Sepharose 2B Sepharose 4B Sepharose 6B 2 4 6 40x I0 20 x 10 4 x I0 20x I0 5 x l0s. I xl0s .Approximate agarose Concentration |%) Approximate exclusion limits (mol. tromraphy 381 $¢eL For completely non-aqueous separations. however unusable with acetone. High rates are permitted by their total rigidity.ene. a gel that will swell in an organic Ivent is required. e 11. trichlorobenzene. The gel structure is unaffected by mperatures as high as 150°C. perchloroethylene. These fine glass spheres are manufactured from osillcate glass to contain large number of pores within a very narrow size distr ibution. A list of some r epresentatives ithis material are listed in Table 11. It is a rigid cross linked pol ystyrene gel which be prepared in a range of different porosities.10 Commonly Used Porous Glass Beads xeluslon limit CMol. These glass spheres h ave a lecular exclusion limit ranging from 3000 to 9 million daltons. alcohols. Conto|led pore gl. dimethylsulphoxide chloroform .) * Bio-Glas 200 i Bio-Glas 500 Bio-Glas I000 Bio-Glas 2500 lxI0 4x 10 I x l0s 4x I@ . The gel can be used with such solvents as tetrahydrof uran. Styragel provides this option. or water . The g lass beads are ated with hexamethyldisilazane to circumvent this problem. suffer from a iortcoming of adsorbing a significant amount of protein on their surfaces. These glass beads. carbon trachloride and others. It is. Wt. cresol.10. however. .s lad. The eluant is steadily added and the e ffluent in various fractions th be analysed. need not be hydrated (swollen) at all. Although the initial swelling is fai rly rapid. a very high molecular weight times to help in the calculation of the void volume. the ge l to the swollen form.*Manufactured by Bio-Rad Laboratories Gel permeation chromatography can be performed either by column or thin layer Column preparation. Blue dextran. A knowledge effluent volume of a particular compound is useful for the calculation of its di stribution which might be useful for molecular weight determination. Thus. The swelling time be reduced drastically by heating the gel slurry on a boiling water bath for 1-5 hours. on the gel type (the higher the gel porosity. . whereas overnight swelling is enough for Sephadex G-200 should be allowed to swell for three days. Air bubbles must be removed the column to a vacuum pump and the level of the liquid must never be allowed lower than the top of the bed. Elution is usually car ried constant hydrostatic pressure head to achieve a constant flow rate. to reach equilibrium. The gel bed is supported in the column on a glass wool plug or nylon net and the previously the form of a slurry and allowed to settle. Po rous on the other hand. This may be done by allowing a known weight of the gel either in water or in a weak salt solution. The greater the porosity. the taken to attain equilibrium may be longer. The general principles of column preparation are the same as previously under the section on chromatographic techniques. the more wi ll be the required. Sample is applied in a manner indicated previousl y (see The volume of sample that should be applied varies as per column size and the type of the gel used. the greater the time required). Prior to use. a layer of hydrated gel is applied to the plate. Gel permeation depends only on the molecular sizes of the macromolecules . refracti ve index. ionic strength. Equilibrium must be carried out for at least 12 hours. For use. ionic strength and other parameters. TLG has found numerous applications in clinical immunology and immunochemistry. lactate dehydrogenase. In this technique. methyl esterase. The applied either as a spot or as a band. Determann. 1. thin layer gel illtration (TLG) is ideal since very small sample volume is required for technique. T here a fixative to adhere the gel beads. The plate is not dried at all and is placed in an contai/ler at an angle of 20°. radioactivity is measured. sir Detection. and polyphenoloxidase for size heterogeneity st udies. Such equil ibrium to normalize the ratio between the stationary and mobile phase volumes. from human granulocytes. The common detection methods include collecting and analysing fr-actio and continuous methods with flow cells in which ultraviolet absorption. and buffer composition. It has the advantages. glyoxylate reductase. peptides and nucleic acids). TLG has been used to study ce rtain such as adenosine deaminase (separation of high and low molecular weight forms). Thin layer gel chromatography.not paralleled by any. The plate may then be developed for a sui table the separated components detected by suitable means. The plate is connected to reservoirs at either end by paper bridges.0ther chromatographic technique. acid oxidase. many cases TLG has been combined advantageously with immunological technique has bee used as screening procedure for several Immunopathological involving a!tered immunoglobulin levels. This is a boon for separation of those macromolecules which are labile under certain conditions of temperature. -glucuronidase from human urine. and Johanson and Rymo (in 1962) showe that gel permeation chromatography could be conducted using thin layers of gel. chromatography therefore can be conducted under virtually any condition of temperature.382 Bphystca Chef. Advantages of Gel Permeation Gel permeation is the gentlest of the chromatographic techniques. and RNA synthetase (all of these molecular weight determination). pH. other enzymes have also been studied using TLG. These macromolecules can . TLG is used mainly for the study of hydrophilic substances which require conditions (proteins. the macromolecule of interest may by providing necessary conditions. 2. Such instability causes large scale changes sizes making them migrate differently from other molecules which ma) molecular weights. Apart from the above named conditions. 3. For reasons which we know nothing about. Other chromatographic techniques do suffer from some Such macromolecules can be separated by gel chromatography is almost nil. macromolecules can be damaged adsorption too. After purification.separated under the conditions where they are stable. there is less zone spreading in chromatography as compared to the other techniques. Conversely. . 4. those macromolecules that can be renatured after they have destabilized may be purified from other macromolecules of similar molecular size s deliberately destabilizing them. One of the advantages of this method of desalting is t hat the macromolecules are eluted with essentially no dilution. it is separately discussed below (also see Box 1 1. their ultimate purification. The method is especially useful for the separation of 4S and 5 S t-RNA. This can be easily performed using gel filtration since the distribution coefficients of salt molecules will be largely different from those of macromolecules. ('I) Dilute solutions of macromolecules with molecular weights higher than th e exclusion limit may be readily concentrated by utilizing the hygroscopic nature of the dry gel. and oligonucleotides. molecules above 5000 daltons will elute very quickly but the molecules less than 1000 daltons will be retarded. Prot eins. Molecular Weight Determination by Gel Filtration Gel chromatographic separations are achieved by means of differential distributi on of sample .made clear that gel filtration has been used for separation of such lo w molecular weight compounds as amino acids. (//) One of the common separation problems in biochemistry is the removal of salts and small molecules from macromolecules. Lest it is thought that the method is only useful for large molecules. the teichoic acids. it should be. usually Gram positive) from the invariable contaminants. It is also the most satisfactory method for separating DNA (from bacteria . in a Sephadex G-25 column. nucleic acids. enzymes. although G-25 is ' preferred for its rapid action. polysaccharides. hormones. This treatment leaves the macromolecular solutio n concentrated but at the same time unaltered in pH or ionic strength. For example. small peptides. Due to the extreme importance of this application. gel permeation chromatography is chiefly used for the purpose of separation of biological molecules leading to. and even viruses have been separated in various experiments which have used different types of ge ls or glass granules. Sephadex G-200 absorbs 20 times its weight of water. (/v) Perhaps one of the most important applications of gel permeation chromat ography is in the determination of molecular weight of macromolecules. antibodies.3).As is evident. in a gel filtration experim ent if one determines the effluent volume one can calculate its distribution coefficient. Since guanldinium chlo ride makes the behave like randomly coiled linear homopolymers. the position of coefficient of the unknown protein on the plot will lead to the determination of its molecular of course. Thus. shapes proteins vvaT widely and may lead to erroneous assignments of molecular weights. This can be obviated by choosing a solvent whose properties are such that it confers identical shapes protein molecules regardless of their original structure. have the same such that their dimensions are a unique function of molecular weight. both standard and known . In most of the cases. The distribution characteristics of a given macromolecule are a funct ion of the size and the hydrodynamic particle formed by the solute molecule. it might be the ideal solvent f or molecular . one presupposes here that all macromolecules. In a previous section relating to the principles of this method we already derived a mathematical relationship between the effluent volume of a par ticular compound its distribution coefficient.between the stationary solvent within the pores of a gel arid the mobile eluting solvent the pores. and the size range of t he gel pores It then logically follows that the distribution coefficient of a solute can be r elated to its molecular a fair degree of certainty. If distribution coefficients of standard of known molecular weight are plotted against the log of their molecular weights . and Mo is the monomer unit molecu lar Viscosity measurements on proteins in 6M guanidinium chloride have related Rg wi th Min following manner R = (constant} M Effective hydrodynamic radius. This c an be to the number of units in a flexible polymer.000 to 80. pH 6.000 to 300. MW = 158. dictates solute partitioning in a gel.000 / " Transferrln (Human). MW= 88. This has been proved experimental ly leading popularization of gel chromatography in 6M guanidinium chloride as a useful meth od of molecular weight determination. the sample is carboxymethylated by addition of iodoacetate and the pH readjusted to Chromatography is carried out and the effluent volume of the desired macromolecu le determined.000 . The sample is prepar ed dissolving it at a concentration of about 1% in 6M-guanidinium chloride and 0.000 I 4"9J "Serum albumin (Bovine). This is used to calculate the distribution coefficient of that molec ule. MW = 67. The gels usually employed for this purpose are 4% (limit of molecular determination I0.2 " Globulin (Hi. It is then a corollary that partit ioning of a polymer is a function of its molecular weight. The sample is then incubated for about 8-10 hours.hydrodynamic properties.6. is of gyration of the polymer. The solvent used is 6M guanldinium chloride in water.384 There is a classical relationship between the average dimensions of flexible lin ear and their molecular weights. 5. M. a is the empirical factor related to a function is related to effective moment of unit length.I 2-mercaptoethanol (mercaptoethanol is used to disrupt disulphide bondsbetween The pH is adjusted to 8. Where Re is the radius of a sphere of equivalent.000 daltons) and 6°/6 (limit of molecular weight 1. Re.000 daltons) agarose.nan). MW = 45.= | Ovalbumln.16 The orrelation of molecular weight and elution volume for proteins from a gel permeation column.000 eC (H . ..0 60 70 80 90 100 I I0 Eluflon volume Figure 11. 4. = . Likewise. Less albumin in blood plasma may indicate liver disease. only the molecular weights of the constituent polypeptide chains of a pr otein can be measured. 1. there are other methods of determining such binding. column calibration curves can be constructed. The positi on of distribution coefficient of unknown polypeptide on such a plot then gives its molecular weigh t. 2.g. Two differ ent techniques may be employed. 3. .plot is shown in Figure 1116. It should. temperature. To be sure. However. Altematively log molecular weight is plotted against elution volume. Special dextran gels have been developed for this purpose. none of the other methods has the advantag e of operating under any given condition of pH. Either one can use a gel permeation column equilibra ted with the small molecule and then passing the protein through it. there are a few other analytical uses that gel permeation can be put to. substrat e : enzyme) can be studied easily with the help of gel permeation chromatography. Analytical Uses of Gel Permeation Apart from determination of molecular weights of macromolecules. Binding affinities of small molecules with macromolecules (e. These give quick and reliable measures of plasma proteins. To determine the molecular weight of the native protein. distribution coefficient. or ionic strength. denaturing condition s of this method. The most popular ty pe of calibration curve is a plot of log molecular weight vs.Cmatography ' 385 distribution coefficient of a number of polypeptide chains of known molecular we ights have been obtained. however. recourse to o ther methods is required. Or one can try separating a small molecule bound to protein by gel permeation. measu rement of different plasma proteins is of great diagnostic value since alterations in thei r concentration may alert the doctor and help him reach a conclusion about the dis ease that the patient suffers from. be pointed out that under the reducing. Such a. A simple calcula tion then is required to determine the binding constant. This can be easily done using gel permeation. .It is necessary during study of RNA metabolism to distinguish various fr actions of RNA. 17 B): . This technique is extremely us eful in the separation of charged compounds (even uncharged molecules can be *charged" by va riance of pH as we will see later).17. The sample contai ning the ionic species to be separated is allowed to percolate through the exchanger for such a length of time as will be sufficient for the following equilibrium to be achieved (Figure 11.17A). which in turn depends mainly on the re lative charge. insoluble support medium. The exchanger is prepared in a way that it is fully charged (Figure 11. The basic process of ion exchange is illustrated in Figure 1 1. and the degree of non-bonding interactions.4. This medium may be covalenfly bound to posit ive (anion exchanger) or negative (cation exchanger) . The value of this technique lies in the fact that cond itions can be so manipulated that some compounds are electrostatically bound to the ion exchanger whereas the others are not. The governing factor in ion cxc ha-ne reactions is the electrostatic force of attraction. Ion exc hange separations are carried out usually in colums packed with an ion exchanger. the radius of the hydrated long. Long bound electro statically to the exchanger are referred to as the counter/ons. The ion exchanger is an inert. Ion exchangers can be divided into two groups: anion exchangers and cation exchangers. ION-EXCHANGE CHROMATOGRAPHY Ion exchange rny be defined as the reversible exchange of Long in solution wtth long electrostaticalty bound to inert support medium.functional groups. or by increasing the pH of the solvent and hence converting X÷ to an uncharged specie (Figure 1 I. Y displaces X÷. (D) F-Jution by Increasbw the concentmn of Y in the. the higher will be the pH required to elute it. 17C.Jchange (exoxnple cation eJchange). X÷ X÷ X÷ X÷ X÷ F/ure 11. X.solvent . (A) Exchange r has been prepared with Y as tlaz cotmtedorc The on to be separated from the samp!e IS X'.opposite charge associated with the exchanger matrix.17 Bascprocess oflon e. The principles discussed above also apply to macromolecules such as proteins and nucleic . the higher the concentration of Y÷ required to elute it. Bound long. D). Concentration of Y÷ required to elute X÷ will depend up on the quantity of charge possessed by X÷. X X xxx y Y y + +. t he rest of the uncharged and like charged species can be washed out of the column. when the pH is being changed. This molecule can now exchange sites with the counterion as shown inthe above relationship. X÷ can now be eluted either by percolating the medium with increasing concentrations ofY÷ (th is increases the possibility that Y+ will replace X+ in the above stated equilibrium because the former is more in concentration|.. and is dlsplcmed. X÷ is the charged molecule (bearing charge s imilar to the counterion) in the sample to be separated. . In the second case. the higher the pK of X÷.is the charged cation exchanger and Y÷ is the counterion of the . (C) Elution by Increasing the pH of the solverrL X÷ is converted to an uncharged species. Once the exchange of counterlon with the sample ion has been achieved. The greater the charge possessed by X÷. The neutral and anionic molecules will not bind at all.E-Y÷+X÷ E-X÷+Y÷ where E. (B) Catlon X+ has bound to column dLsplacing Y'. can be exploited for purification purp oses by using ion exchange chromatography. particularly proteins. These large n]olecules. if chromatogr aphed on a cation exchanger will remove many of the anionic protein species. The macromolecules can. The am photerlc nature of macromolecules. This preparation. anion and cation exchangers since they posse ss both types of charges. then. can bind to both. be made to bear more negative charge s by increasing the pH {resulting in a stronger binding to anion exchanger} or more p ositive charges by reduclng the pH {resulting in a stronger binding to cation exchanger}. the desired protein may be made to beh ave as a ©ation by lowering the pH of the protein mixture {the pH is lowered to a limit whe re most of the other proteins in the mixture behave as anions}. This process w ill remove . howeer. For example.acids. which are capable of possessing both positive and negative charges. {F*) & (G*) are not ex. elute quickly and are discarded. {C-).@ ®@® Performing anlon-exchange chromatography.18 Sequent use of cation and anion exhange chromatoraphy results in a large degree of purtfumflan of the desired protein from a mixture of several . (D-) & |E-) bind to beads and csn be eluted later. Thus the protein solution has been enriched for (D) Ftgure 11. The desired protein {D) is now in a mixture of only three proteins.0 ®. of the protein solution to 8.®@ and so einte out quickly. Two unwanted proteins have been removed and we have a mixture of five proteins. These can be eluted later and collected.0 F & G become positively charged while (C) & (E} assume negative charge Buffer pH 8.snged. Changing the pH. (C÷} (D*) (E÷} (F÷ & (G÷) hind to the bead and are retarded. Increasi ng the cross linkage increases the rigidity. This is better explained by the example in Figure 1 I. reduces porosity. Polystyrene resins are prepared by polymerization reaction of styrene and diviny l benzene-CH = CH2 CH2CHCH2---CH---CH2 + CH2--CH--CH2-CH ffi CH2 CH Diviny Ibenzene Polystyrene The above structure is repeated in three dimensions.Chromatography 389 many of the unwanted proteins from the starting mixture and the resultant mixtur e is rich for the desired protein. 8% cross linkage finds wide'spread use. Selection of one or the ot her type of resin is done on the basis of compounds being separated. and reduces the solubility of . If the preparation is now chromatographed on a n anlori exchanger. A higher concentration of divinylbenzene produces higher cross linkages. If the pH of this resultant mixture is now increased the de sired protein will exist predominantly as an anion (the pH is increased to a limit where most other proteins in the mixture still behave as cations). many cationic protein species will be lost. It should thus be clear t hat anion and cation exchange chromatography used sequentially can afford a large degree of pu rification. with the number of cross li nkages determined by the ratio of divinylbenzene to styrene. and cellulose. reduces swelling. 18. Resins made from both of these materials differ in their flow propert ies. ion accessibility. Types of Ion Exchange Resins Two main groups of materials are used to prepare ion exchange resins: polystyren e. and chemical and mechanical stability. for example. the resin can exchange anions rather than cations. Strong anion exchangers are prepared with a tertiary amine. carboxylate groups can be attached to the aromatic rings instead of sulphonic acid groups. Selection of high or low cross linkage resin depends up on the compound being separated. nearly every aromatic nucleus. a re not free to leave the resin unless replaced by other positive long. To prepare weakly acidic (weak cationic exchangers) exchanger. depends upon the strength of the acid. These protons. The exchange distribution fhnction. Resins substituted with sulphonic acid groups are strong cationic exchangers. by sulphonation in which a sulphonic acid group is attached to. If basic functional groups are introduced. however. yielding a strongly basic . however.the polymeric structure. Acidic functional groups are easily introduced. Weak acid groups usually result in high capacity. Sulphonic ac ids are strong acids with essentially completely dissociated protons. The total number of equi valents of replaceable protons per unit volume of resin determines the exvhan capac of the resin. yielding a weakly basic tertiary amine. Weak anionic exchanger can be prepared with secondar y amines. Carboxymethylcellulose' {CM-cellulose. The unsatisfactory separation of macromolecules on polystyrene type of resins pr ompted development of the cellulose-based exchangers. unsatisfactory when macromolecular separations are to be carried o ut. Strong cationic cellulose exchangers {sulphoethyl} and strong anionic exchangers . They are. Cellulose is a high molecular weight compound which can be readily obtained in a highly pure state from such common raw materials as cotton.ealdy acidic. Cellulose resins have much greate r permeability to macromolecular polyelectrolytes and possess a much lower charge density as co mpared to polystyrene exchangers. w. softwo od. and hardwood. weakly basic. Polystyrene resins are very useful for separating small molecular weight compoun ds. and DEAE cellulose CHOCH2CHN {CH2CH3}. A list of a few common resins is given in Table 11:11.Dowex 50 Amberlite IR 120 SP-Sephadex Amberlite IRC 50 CM-Sephadex Dowex 1 Amberlite IRA 400 QAE-Sephadex Dowex 3 Amberlite IR 45 DEAE-Sephadex DEAE-Sepharose 390 Biophysical Chemistry quarternary ammonium group. however. anionic exchanger} are examples of main derivatives of p ractical value. cationic exchan ger} where the CHOH group is converted to CH2OCH2COOH. It is logical then. Both Se phadex and sepharose types are particularly valuable for the separation of high molecular w eight proteins and nucleic acids.{guanidoethyl} are also available.11. the modified cellulose is known as microgranular c ellulose. Thus. Table 1 I. does not give high flow rate. Typical examples of cellulose based ion excha ngers are given in Table 11. It. Other alternative resins in use are derivatives of cross-linked agarose.styreri with -CH2NMe3CI Diethyl {2 hydroxypropyl} quarternary amino cellulose Polystyrene with secondary amine Diethylaminoethyl cellulose Diethylaminoethyl agarose Cellulose may be treated in such a way as to remove the amorphous portion. Chromatographic papers are based on cellulose. however. rather than being fibrous in nature. 11 Select lon Exchange Resins Nature Trade Name Strong cation Sulphonated polystyrene Weak cation Strong anion Weak anion Sulphopropyl cellulose Condensed acrylic acid Carboxymethyl cellulose Poly. Each form is commercially available in gel and bead forms wh ich possess good flow and exchange properties. that if the c . This modified cellulose has even better resolution than the natural fibrous cellulose . These papers are commercially available. and aminoethylcellulose (weak anion exchanger). . one can carry out ion exchange chromato graphy on paper also.ellulose of the paper is modified to DEAE-cellulose. cell-ulose citrate (weak cation exchanger). Other modified papers inclu de cellulose phosphate (strong cation exchanger). Cross-section of such a swollen cation exchang e bead is shown in Figure 11. H÷ is the (0. which results in the burial of many ch arged functional groups which are then unavailable for exchange. Swelling makes these fun ctional charged groups to become exposed.5N NaOH}. ff impurities like metal long are present. 19 Cross-section of a suxlen cation exchange bead with the functiona l charged groups exposed. Exactly the reverse is the case wit h cationic exchangers. the matrix can b e treated with a chelator such as EDTA. This is also known as precycling. the slow sedimenting material (fines) is decanted. It is necessary to remove fines since the presence of a large number of such fines wil l result in a decreased flow rate and unsatisfactory resolution.hromatography ' 391 Prepmtion of the Ezchene Medium Conversion of the exchanger from the form in which it is supplied to the form in which it is to be used is known as the preparation of the exchange medium and is essential for satisfactory performance of ion exchange chromatography. Finally. (//) Removal of very small particles of the exchanger (fi nes). This is necessary since t he dry exchanger contains densely packed polymers. (i)Swelling the medium. the exchanger is repeatedly suspended in a large volume of water and after the large r polymers have settled down.steps that are of absolute importance in exchanger preparation. Apart from remo ving the impurities there are three other..19. Such fines are usually genera ted during swelling ff the exchanger is subjected to vigorous washing and stirring. To remo ve fines.5N HCI) and then with a base (0. Swelling of anion exchangers is usually carried out by tre ating it first with an acid Figure 1 I. (//0 Final ly the exchanger . Choice of Buffers The choice of buffers. in case the counterion to be introduced is Na÷. formic acid if formate is the counterion. This is accomplished by w ashing the exchanger with different reagents depending upon the desired counterlon to be in troduced (NaOH. excess counterions are removed by washing the exchanger with large volumes of water or dilute buffers.). Following conversion of the exchanger to the desired form. NaNO3 ff NO is the counterion. Anion ."has to be equilibrated with the suitable counteri0ns.etc. is dictated by the c ompounds to be separated and the type of ion exchange being carried out {anionic or catio nic}. which maintain the pH of the column. HCI ff H÷ is the counterion. The pH of the buffer should impart the same charge t o the sample long as is present on the counterion. A llst of volatile buffers used in ion exc hange chromatography is given in Table 11.12 Some Volatile BuYers Used in lonExchange Chromatography Buffer pH range Ammonium acetate Ammonium formate Pyridinium formate Pyridinium acetate Ammonium carbonate 4-6 3-5 3-6 4-6 8. Table 11. The pK of the buffer should be as near as possible to the pH at which the system is buffered.I0 .12. the buffer long will indulge in ion exchange and hamper sample component exchange.392 Biophysical Chemistry exchange chromatography should be carried out with cationic buffers. This results in high buffer capacity which can withstand the local changes of pH in the column easily. If anion exchange or cation exchange is carried out wit h anionic or cationic buffers respectively. Reverse is true for cation exchange chromatography. pyridine. use of a long column might in fac t be beneficial. they will have enough opportunity to diffuse and therefore will be eluted in a l arge volume leading to peak broadening. Conversely. might impose a peak broadening effect. whereas columns long and narrow. if the elution is being done with a solutio n of one concentration. Thus. Samples exhibiting stability over a wide range ofpH may be separated using either type of the exchanger. the exchanger selected should operate within this ra nge. The pH of the buffer used is usually maintained at about one pH unit more or les s than the isoionic point of the sample components. and alkylamines are used when anion exchange chromatography is being performed. This isso since in a long column. their molecular weight and the specific requirements of t he separation. However. cationic buffers such as Tris. the diameter of the pores is usually controlled and some molecular si eving effects might also play a role in separation. for a sample exhibiting more stability above its isoionic point. especially proteins. The degree of cross linkage should therefore be fixed taking into account the molecular weight s of the sample components. The columns used also have an effect on the resolution obtained. . the free long have to traverse a long way down before the fractions are collected. ff the sample is more stable at a pH below its isoionic point. use of a cation exchanger is advocated. Since cross linked gel material is used as an exchanger. Many biological components. during elution. are stable only wit hin a fairly narrow pH range. Therefore. might have an effect on the exchanger capacity. Thus. I n the long column. the cross linkage. although it might not affect the ion exchange mechanism as such. The volume of exchanger used for separation is usually 2-5 folds greater than that needed to bind all of the sample. However. an anion exchanger might be more useful. rather than a gradient elution. excesses greater than this are avoided. General rules could be formulated for the choice of exchanger depending upon the stabili ty of the components to be separated. Generally.Practical Procedure We have already stated that the choice of the ion exchanger depends upon the sta bility of the sample components. Columns of a hi gh diameter to height ratio usually give a better resolution. When . lleatlon Ion exchange chromatography has several applications some of which are listed be low. Initial buffer pH and ionic strength should be adjusted so as to just allow the binding of sample components to the exchanger. and phosphate are used. .Chromatography 393 cation exchange chromatography is to be done. b arbiturate. (/) Perhaps the most stimulating use of ion exchange chromatography is in am ino acid analysis. The latter gives a better resolution. This significant advance in instrumentation did wonders for protein chemistry (s ee Box 11. In fact the amino acid "autoanalyser" is based on ion exchange princip le. If isocratic separation is to be performe d.4). the sample volume is about 1-5% of the bed volume. During a nion exchange |the pH gradient decreases and the ionic strength increases. anionic buffers such as acetate. Sample volume is not an important consid eration when gradient elution is to be performed. i Gradient elution is more commonly performed than Isocratic elution. Gradient e lution |could be stepwise or continuous. Size of the column and the capacity of the exchanger are the two factors which d ictate the amount of sample which can be applied. On the other hand d uring cation exchange both pH and the ionic strength increase. magnesium.. other biological amines. Rather than complete deionisation. in the eluent is considerably higher than that in the sample. metal ion free reagents are is commercially performed by ion exchange chromatography. Complete of water or non-electrolyte solution is performed by exchanging solute cations hydrogen long and solute anions . be "read" by atomic emission or absorption spectrometry and the results to the original concentration in the sample. The mixture of nucleotides as a result of treatment with DNAses and can be readily separated by ion exchange chromatography. (v) There are many situations where is below the limit detected by atomic emission or atomic absorption spectrometry .for hydroxyl long. are exchanged for sodium. These long can now be eluted. enough trace metal long concentrated over the exchanger. (v0 Apart from the above applications. (///) This is the most often quoted application of ion exchange. since it is u sed all laboratories as fast and effective method of water puriflcatlon. and Chargaff used this technique for the purpose. (iv) For many biological applications. The same method is also used f softening of drinking water. all multiple cations like calcium.394 Biophysical Chemistry (//) Ion exchange has been extensively used to determine the base composition acids. These samples are passed over an ion exchange resin that holds the metal enough sample has been passed over the resin. . and organic acids and bases. etc. iron. This is usually water in a mixed bed of anion and cation exchanger. Throughout which established the equivalence of adenine and thymine. ultrapure. ion exchange chromatography has been used the separations of many vitamins. . known as receptor. a given hormone will bind to only a s pecific glycoprotein.g. Buffer Protein mixture layered at the top of the column obd column ] I. Let the substrate analogue (molecule re sembling the substrate but not capable of reaction] specific for this enzyme is coupled to a column matrix which is immobile (e.. LLLLL] [LLLLLL [LLLLLU LI.20]. All other molecules. AFFINITY CHROMATOGRAPHY As opposed to all the chromatographic procedures described thus far which exploi ted small physicochemical differences of molecules in a mixture for their eventual s eparation. Let us suppose that an enzyme is to be purified from a mixture of thousands of proteins.396 5. will be retarded by the matrix. noncovalent b inding of other molecules called ligands. agarose). which have no specificity for the ligand wil l pass down and out of the column. a given antibody will s pecifically bind to only a given antigen and not to others. This a mazing biospecificity is not limited only to erymes. the conditions of the wash solution are altered so that the enzyme dissociates from the ligand and elutes from the column./aity chromatography exploits the capacity of biomolecules for specific. When the mixture of proteins containing the e nzyme to be purified is allowed to percolate through such a column. only the desired enzyme. a. largely in the purified form (Figure I 1. After all the undesired components have been flushed out of t he column. LL . which is capable of binding specifically to the immobilized ligand (substrate analogue). Any student of biochemistry will be familiar with the concept that a given enzym e will bind and react'with only a group of substrates and will not react with others. situaten the plasma membrane surface. L.L L I. one can is olate the corresponding antibody. one may be ab le to purify its corresponding receptor. and even whole cells and cell fragments. 13. macromolecules other than enzymes are also separable by affinity principle. Isolation of messenger RNA from a crude RNA preparation is one of its often used special applications.characteristic ligand. A short list of separations c arried out employing affinity chromatography is provided in Table I I. thus. affinity chromatography exploits the biological affinity of the macromolec ule for its . it may be said that all such biomolecules involved in specific interaction with other m olecules can be purified with the help of affinity chromatography. if the ligand immobilized is an antigen.-. In short. it is currently used to purify nucleic acids. or if the ligand immobilized is a hormone.Proteins not specific for the ligand elute out Buffer .L C]anging the Composition of eluttng buffer: The desired protein elutes out Ftgure 11.LLL. Affinity chromatography is not limited to proteins only. . ---.L. As said earlier.20 Principle of affinity chromatography Thus. Concanavalln A The picture of affinity chromatography presented in the above discussion would m ake one that the desired macromolecule will be totally purified in one single step.. which interact wit h more macromolecule present in a given mixture. Most such complications arise because of nonspecific of sample components other than the desired one on to the matrix.g. or ionic . pH. This comp lication . For example. . Usually ionic hydrophobic interactions are involved in such non-specific adsorption. which is to be isolated. In practice. Glyc01iplds Fat Cells mocyUn Tryptophan Benzaxnidine Heparln Antibody Poly (U) or Poly dT .Specific Ligands Commonly Used in Chromatography Macromolecule/Cell Llgaud Avdm a-Chymotrypsin Thrombin Coagulation factor Interferon Poly (A] messenger RNA Ribosomal RNA Glycoprotelns. Another type of complication arises when one uses ligands. Lyslne Concanavalin A Insulin. many dehydrogenases rather than the specific one. when a coenzyme like NAD÷ used as a ligand. Howe ver. a number of complications of th is picture are always present. be taken care of by judicious choice of operating conditions (e. temper ature. it is an picture of affinity chromatography.. Before initiating specific laboratory procedures for affinity chromatography. and the means ofcovalenfly binding it to the matrix. This allows flexibilit y in the design of experiments. affinity remains probably the most powerful tool for purification of biomolecules and an amazing degree of purity impossible to attain by other procedures. ionic str ength. A general discussion of these points is provided below. {iv) It should contain large numbers of suitable chemical groups for ligand atta chment. its nature. (/0 It should possess good flow properties. is required. (it0 It should be chemically and mechanically stable at varying pH. Major variables. are (0 The type of matrix used. s ome '. and denaturatlng conditions employed for binding and elution. and (///} The conditions exploited to bind and dissociate (elute) the macromolecul e from the column.to the ligand-matrix conjugate. They are discussed below (0 The matrix should be inert to other molecules to minimize non-specific adsorp tion. matrix Characteristics desired of an ideal insoluble matrix for affinity chromatography have much common with those desired for gel filtration. Inspite of all these complications. . which need careful consideration. (//) Selection of the ligand. In such cases it becomes necessary to use specif ic procedures to effect the desired separation. change in pH. used ones are agarose. Their most serious drawback is a high degree of nonspecifi c adsorption. be done in such a way as to no t to impair the binding of the ligand with the desired macromolecule. the conditions are so arranged that the enzyme substrate re action not occur. antigen epitopes. synthetic agonists. polyacrylamide. suffer from a major disadvantage. particles which are uniform. are the usual candidates. pore glass beads provide mechanical rigidity and chemical inertness in addition to very good flow rates. receptor antago nists. Under certain circumstances. these might be achieved by omission of required metal long. however. or omission of the second substrate if the enzyme catalyzes a bi-molecular reaction . however. Of t hese agarose beads are by far the most often used because they possess most of the cited above. The selected ligand should meet two most important requirements. High porosity provides a large su rface area attachment of the ligand and allows better interaction of the desired with the immobilized ligand. This problem can be circumvented to some extent by treatment hexamethyldisilazane. The linkage modification should. these circumstances. .. and controlled porosity glass beads. In practice. spherical. The interaction. and rigid are used. undesirable trait is heightened even further when they are substituted by ligand s. however. etc. they suffer from a lack o f porosity. it might be the onl y choice. they to contraction when denaturant solutions are possess many of the desirable criteria outlined above. (t) The ligand interact strongly with the desired macromolecule. Agarose beads. (to The ligand to be bound possess functional groups that can be modified to form covalent linkage with the matrix. Controlled pore glass beads might very well be the matrix of future. Substrate analogues. effectors. Llgand Selection The ligand to be used in construction of an affinity column is selected after consideration. It is not usual to select a substrate as a ligand. must however not be strong. A very strong affinity will require a drastic treatment to dissociate th e complex elution which might damage the desired macromolecule.298 Boptu3scol (v) It should preferably be highly porous. enzyme cofactors. The activated suspension is nowwashed with about 20 times the gel volume with a buffer [buffer s such as . The pH is maintained by the addition o f 2M NaOH. To maintain the pH at 11. the support matrix must be washed repeatedly. After the reactions establishing the linkage are over. For lesser activation a lower amount of cyanogen bromide suffices. Usually just 10-15 minutes are required for the reaction to be completed. Usually 300 mg of powdered cyanogen bromide is used per ml of packed gel gives t he maximum substitution. The most common method of activation of polysaccharide supports (agarose) involv es treatment with cyanogen bromide at alkaline pH (pH = 11. The chemical methods used should be mild so that the ligand or the matrix Is not damaged. The reacUon conditio ns and the relative proportions of the reagents determine the extent of activation of the m atrix particles. The pH of the gel suspension is raised to 11 and all the cyanogen bromide powder added at once . The activation reaction (Figure 11. the entire reaction should be carried out in a fume hood. Due to the toxicity of cyanogen bromide. and (/0 covalent attachment of t he ligand to these activated groups. The reaction is exothermic and therefore there is a need to maintain the temperature constant at 2O0 at all Umes. Moreover the reacUon gener ates protons decreasing the pH.21A] begins.0).Ligand Attachment Covalent coupling of the ligand to the supporting matrix involve the following s teps: (/} activation of the matrix functional groups. the mixture is continuously stirred and an electrode dipped into it at'all times. CNBr activated poly saccharide apports which are freeze dried are commercially available. 14 summar/z methods of activation other than CNBr method.5-10.g11. Table I. It is known from chemical and spectroscopic evidences that activation of polysaccharide apports by CNBr gives rise to the reactive iminocarbonate structures(Figure 11.pH-.21 (A) Activation of polysaccharlde support with CNBn and (B) Coupling of amlno llgands.tion with Ligand coupling type of Iigand Reacts with amines And other nucleophiles Reacts with amino groups and other nucleophiles . -CH HO "0 + Br---C -= N Cyanogen OH ' bromide Iminocarbonate ljure 11. glycine should be avoided because amino groups compete wi th the gand being coupled] at a pH of 9. I I.2 1A). 14 Alternative Methods of Activation of Polysaccharlde Supports Active functionality for Reac. ammonium acetate.0 Epoxides Periodate hro raphy 399 rls. Sodium bicarbonate and borate buffers are the usual liolce. 0. pH suspension is gently stirred overnight in a cold room or for a couple of hours a t room During this time the ligand is covalenfly attached to the support medium as in Figure 11.2 IB.CHO Reacts with amines or hydrazines The requisite ligand may be attached to the matrix immediately after the activat ed suport been washed. It is likely that the ligand is attached through an isourea l inkage.g.. Coupling of amino-containing llgand to CNBr activated support is nr mally suspending the support and the ligand in a basic buffer solution at pH 9 (usuall y is the same as that used for washing the activated support.I -. .25M NaHCOa . The pH sensitive and therefore the pH must be maintained around 9. e.0 all the time. 3. Examples include hexamethylenediamine.aminohexanoic acid. The procedures involved in attaching the ligand to the spacer arm frequently involve the use succinic anhydride and a water soluble carbodilmide. the matrix is washed with O. Chromatography Procedure .6 . The rrmcromoleade binds without any sterlr hlndmnce. and 1. (B) llgand wtth a spac arm.1M solution ofpH 9.diaminohexane. nmcronlecule cannot attach due to steric h6ndrance. Due to the ligand is projected at a distance from the matrix and the desired macromolec ule will bind it without facing any steric hindrance (Figure 11.22(A} shows that if a ligand is directly attached to activated groups of the the macromolecules might encounter steric restrictions due to which they might adsorbed to the matrix. This spacer is known as the arm. Gels attached with differen t arms commercially available.22 Need of a spacer arm. It Is useful to determine the number of ligand groups bound to the matrix as us how useful will the eolurrm be in separation of the desired macromoleeule. A large variety of spac er investigated. it is usual to introduce a s pacer activated groups of the support and the ligand. Th is can be eas.0 This treatment assures destruction of any extra activated groups. number of llgand groups bound is usually expressed in terms of capacity per ml o f matrix rather than in terms of its dry weight. (A) Ligand without a spacer arm is closely he ld to the matr particle.fly performed if a radioactive ligand of a low but known specific act ivity is used.(2.3-epoxypropoxy) spacer arms must possess two functional groups. Figure I 1.3' 1.4-bis . Figure 11.22(B). 6 . To obviate this difficulty.400 Btophyscca After the reaction is over. one to react with the functional groups matrix and the other to which the ligand could be attached easily. Following this the extensively washed to remove any unboand llgand. The procedure for affinity chromatography has many similarities to other forms o f liquid chomatography. the column is eluted with more buffer to remove non specifically bound unwanted molecules. The buffer should also possess a high ionic strength so as to minimize non-specific polyelectrolyte ads orption onto charged groups in the llgand. The purified. N on-speeific elution is . The buffer which encourages adsorption of the desi red molecule on the gel surface is used. bound component may now be eluted (diss. oeiated) by taking recourse to either specific or non-spectfic elution. metal long) required for llgand-macromolecule interaction. The buffer chosen must be supplemented with any cofa ctors (example. The sample is applied at the top of the column and the buffer flow started. Once the macromolecule is bound. The gel beads are swollen much in the same way as described for g el permation before loading onto a column. other contaminants will soon clog the column rendering it useless. These mus t be removed (usually dialysis for a long time) to bring the macromolecule in its native conf ormation. a psychotroplc drug binds to specific receptors on neurons. Affinity separation still remains the most power ful technique for protein separations and hence its increasing use.. The given protein has to be purified to quite an extent before it can be applied to affinity columns. On the basis of the foregoing. This problem has been addressed in several ways in recent years allo wing affinity principle to be utilized right in the initial stages of protein purification. This problem .A brief description of thes . The purified material is eventually recovered in a buffer solutio n which may be contaminated with specific eluting agents or high salt concentrations. the technique suf fers from two drawbacks. This is so because most of the biotechnological applications involve e ither use or production of proteins/enzymes. It is exceedingly costly.C ocjraphy 401 carried out by changing either the pH or the ionic strength of the buffer. The ligands used here are quite costly and the incubation process also is fairly long leading to a further increase in costs. the use of alrmity chromatography is incr easingly required. The other problem isthat affinity chromatography cannot be used as a first proce dure. If affinity is used as a first step. Variations of Affinity Technique With biotechnology coming centrestage. * Morphine. design a procedure to purify these receptors. However.cannot be solved unless an economy of scale is achieved by more frequent use of these ligands. In a book of this size it is impossible to discuss these variations in detail. Chang e in any of these two parameters causes destabilization of the ligand-macromolecule link and cause s the macromolecule to separate and elute out of the column. Specific or affinity elut ion is carried out by (/} addition of compounds for which the macromolecule has more affinity. The high cost is mainly attributable to the process of conjugation of the ligand to the matrix. or (//) by addition of compounds for which the ligand has more affinity than it has for the desired macromolecule. A simple change in conditions like the pH. The faster flow rate also enhances and greatly improves al l the steps of the protocol : washing. ionic strength. Affinity Precipitation The technique has evolved mainly due to the development of what have come to be called .' What is smart about these polymers is that they can exist b oth in solution and suspension (precipitate] form depending on the environment. or polytetrafluoroethylene. Later the protei n can be released from the ligand by a change of buffer. Affinity Cross Flow Ultraflltration " Membranes used here are mostly microporous and are usually made up of cellulose acetate.available on which the ligands may be bound. The pore size is usually 3-5 ram. polyvinylidene fluoride. The prot ein sticks by . temperature. or even the addition of a metal ion ca n bring the polymer down from a solution form to a fairly compact precipitate form. Preprepared activated membranes ha ve also become commercially available. When the whole mixture containing the protein of interest is incubated with it the membrane retains the protein-ligand complex while the contaminants pass through the membrane pores.e techniques is being given with a good list of relevant references at the end so that the desir ous student can supplement his knowledge. The ligand is bound to a ch osen smart polymer and this whole complexis allowed to exist in solution form. Functional groups are made . The flow rates can be very high due to the microporous membrane.the 'smartpohmers. and regeneration. Imagine the ease of purification by this method now. The ligand used must be ve ry specific and th membrane should be of a material that shows very little non-specific adsorpti on. now dumped into this solution. elution. The mi xture from which the protein is to be separated is. This is not all. Once this has happened.5. The column is not present.5. However well you may attach the ligand. Matrixless Affinity Separations There is another variation of this technique which is quite interesting. At this pH chitosan exists in the solution form. pH 5. one of the above named condition is changed and the protein-ligand complex is made to precipitate . The wheat germ lectin has an affinity for N-acetylglucosamine. the cost of affinity chromatography is high mainly owing to the costly ligand as wel l as the long incubation period required to attach this ligand to the matrix. Chitin can be partially deacetylated to give rise to what is called chitosan. After this a simp le gel filtration step can separate chitosan from lectin. The variation that we are going to talk about does away with the above problems by simply doing away with the ligand itselfl Let us understand this variation with the hel p of an example.402 Biophysical Chemistry affinity to the ligand which is in solution form. no rigid matrix is present either. Conventionally. There is no danger of any clogging and the affinity principle can be used right as the first step of p urification shortening the time and cost of purifying the protein of interest. it also has an affi nity for wheat germ lectin.5 at which the chitosan-wheat germ lectin complex precipitate s. As stat ed above. After sufficient time has been given for incubation. Later. Poly N-acetylglucosamine is a natural component o f the exoskeleton of arthropods. the target molecule is recovered from the polymer by changing the buffer. No ligand therefore is required to be covalently linked to this pol ymer for wheat germ separation. The strategy is then to incubate wheat germ extract with chitos an at. Chitosan can act as a smart polymer. In fact. It is easier to separate this precipitate from the mixture now. o ne can try separating wheat germ lectin on an affinity matrix to which N-acetylglucosam ine has been covalently linked as a ligand. pH is changed to 8. some of the ligand leaches off the matrix. The precipitate is separated easily and redissolved by lowering the pH to 5. USE OF AFFINITY CHROMATOGRAPHY IN MOLECULAR BIOLOGY Purification of mRNA . What is more. The kit provides the eluting buffers. molecular biologists frequently have to isola te mRNA from a total RNA preparation which contains other types of RNA. The column is washed twice again by iilling the syringe with buffer and pushing the plunger .In order to study gene expression. Other RNA molecules do not have such poly A tails.. through complementarity. The oligo dT attached matrix comes packed in a 1 ml syringe. The liquid coming out of the syringe contains the RNA molecules other than mRNA. After this the eluting buffer is dispensed at the top of the syringe and the plunger is pushed again. Affinity chromatography is routinely used for this purpose. The entire procedure takes barely 10 minutes. After this one fixes the plunger at the top of the syringe colum n and pushes. viz. rRNA. only mRNA is retarded while the other RNA molecules elute o ut. After other RNA molec ules have eluted out. The eluting liquid contains mRNA which can be collected. Total RNA preparation is layered a t the top of the gyringe column. A few years back a company called Stratagene came up with what is known as a pus h column. and tRNA. recognize and bind to the oligo dT molecules immobilize d on the matrix. This becomes possible because most mRNA molecules have a poly A tail at their 3'-ends . This syring e is the column. These tails. mRNA molecules bound to the matrix are eluted by changing the wash c onditions. Oligo dT (many units of deoxy thymldylllc acid) is immobilized on an agarose matrix. . When total RNA preparation is allowed to percolate through this column. most transcr iption factors constitute just about 0. Kadonaga and Tjian decided to exploit the high affinity of this protein to the GC box sequence GGGCGG.001% of total ceil protein. To give you an idea how low the concentrations may be. The protein was called Sp I and the sequence was called GC box owing to the fact that it was formed entirely of the two nucleotid es. it is necessary that they be purified. (/) Affinity chromatography has been used to purify a large variety of macro . the pr otein recovered after two cycles of DNA-affmity chromatography contained only Sp 1. A crude nuclear extract was passed through a column consisting of these b eads coupled to the oligonucleotide sequence. for quite a length of time. These researchers found that one of the sequences occurring in the regulatory region ( promoter) of a given gene bound to a protein specifically. The column was then washed to remove non-specif ically binding proteins. To study the mechanism of action of transcription factors. This resulted in the elution of the Sp 1.Chromatography 403 Isolation of DNA-binding Proteins A transcription factor is a protein that recognizes ttnd binds to a specific DNA sequence lying in the regulatory region of a given gene. All DNA binding proteins which show sequence specifici ty cn be purified by this technique. They also demonstrated that the binding of Sp I to the GC box stimulated the transcription of the gene.. However. In the 1980s Tjian and Kadonaga were working with the monkey virus SV40. A further gel electrophoresis experiment proved that while the nuclear extract contained thousands of different proteins. ppllcations . Since then several transcription factors have been purified using DNA-affinity chromatography. This binding may promot e the transcription of the gigen gene. They sy nthesized oligonucleotides containing multiple repeats of the above sequence and coupled t hem to solid beads. scientists could not succ eed in purifying these proteins owing to the fact that these proteins are present in extremely lo w concentrations in the ceil. The problem was to purify this protein in order to study it.Conventional biochemical tech niques cannot purify such proteins. Finally the beads were washed with high salt buffer which disrupted th e Sp I binding to DNA. The application of DNA-affinity chromatography is not limited to purification of transcription factors. immunoglobulins. a mixture of ceils when passed through these columns comes out of the column deficient in fat ceils which become bound to the matrix due to insulin. An aft'unity matrix is designed which has i nsulin as the immobilized ligand. lymph node cells. Affinity fractionat ion has an advantage over other conventional types of fractionation (centrifugation etc. nucleic acids. These proteins might. ('t//) Metal chelate affmlty chromatography is a logical extension of the basic technique. and the cells remain viable.molecules such as enzymes. let us consider the case of fat cells. Many proteins which have similar molecular weights and even isoelectric points c annot be separated even by such high resolution techniques as gel filtration chromatog raphy. and even polysaccharides. spleen ceils. This property may then be exploited if the particular metal ion is immobilized o n a gel . and chick embryo neural cells. T and B lymphocytes. however. differ in their metal bindin g abilities. or electrofocussing. oocytes. Many other types of ceils cited above are fractionated by immobilize d antibodies for which their membranes are antigenic. the fractionation is carried out under physiological conditions. without involvement of much trauma. micrometer). membrane receptors. Fat ceils have a large number of insulin receptors on their plasma membrane (about 10 receptors/sq. To illustrate the technique in brief. Cells separated by using alllnity frac tionation include fat ceils.). (/0 Whole ceils have been purified using this technique. temaflvely eflon c be done by cludg EA (which . Hg=÷. However. hepafls B surface gen. Some of em e as foows. e sple is apped at neu pH d e compod eluted by reducg e pH d ioc seng of e buffer. when used as a gd. ese colus e extensively used duses because of e obous advmges. me s ubsces wch have been ped usg s stem e as foows: DNA lerase. () Use of maetic gel beads is oer eension of chromatoaphy. 1. utee es&d receptor. oy a few componen of e ce eact bd to it. Cd=÷. e ure is en passed roll a malefic field. DNA polerases. Blood coagaflon factors so bd to such colums. bosomes.BiophsicaZ Cheraistr matrix by chelation. ne coagenaae. ases. . however. d ate reducmses. etc. (v) Iobed ees is oer ension of e i pciple. ees hang a gh for nucleofldes bd to e colu. Cu+. He-se. e cells suspension e owed to interact e crosph eres.as a good met chelator detely have better for e oh met ion e macromolecule ) in e buffer. lyn-. e oer cells. scffic polucleotide sequences covalenfly nked to agose may be used to pur scHpflon factors d oer DNA-bdg proteus. Eples of such ees e e dehydrogenases . Sly. have a core made up of FeO (maec] d e checly coupled to a prote gd. e meod ves a roue puH of about 95%. e ees may be atmch to gel beads which e packed in a colu. e desed cells e thus purified d c be coect by simply remog e magenflc field. ere is so dence at some cases e obed ees e cannery more acve e ee cotes. Binding of proteins to such metaJ long as Zn=÷. e subsates a stable reaction ure c now be conuy passed roll one end of e colu d e product removed from e oer end. Cron-bl-ose. d Ni=* is pH dependent. . 3. usury polyaclde or aglow. less prone to protea digestion d easily stored as comp to e ee ees. It teracts a lge numr ofblo com nents. e not ected by e maec field as ey do not teract e mec crospheres. scussed above. e des ces (which have become atta ched to e malefic crospheres) move towds e poles of e manet. Sd -d Syste Ce ma-gd systems have become stdd for sepag cen es of macromolecules shang a coon stcturaHfuncon chactestic. Iunoobu negave oces d neoblastoma ces have en ped by is me. Hep is a nat coult found e blood. ogo-dT colums e used for pcaon of A. Iobed ees (so o as sod-ste ees) e o to be more ey sble. l of these e erciy avable too. Cibacron blue is a dye at sctuy resembles nucleodes spite of e fact at it is f more complex en s dye is used as a gd. Co=*. ese gels beads. Poly-U cols ma y so be used stead of oligo-. It has n ensively used fly pucaflon.ong e subsces at have been pued s system e m d e prote plasogen. 5. Lyse-agoseds notbd to algenumberofsubsces. s-ose.4. te-A-. derived from ce w of Sphyloccus aureus bds we to e F reDon of e oobu G. . is prote. a t higher pressures their structure is affected thereby causing flow rate anomalies.perlormance liquid chromatography is a product of the scientific effort towar d optimization of the conventional column chromatography. Ordinarily. The flow rate ther efore is high and the experimental time is shortened considerably. which reduces the time that a solute sp ends in the column. It is routinely used to iso late glycoproteins and polysaccharides. The lateral diffusion is less because of two f actors. Lectins are compounds that bind to a variety of polysaccharidc s"or glycoproteins. the plate number is reduced. The technique may be used with vanishingly smal l amounts of sample (pico or even femto gram). The most used lectin is Concanavalin A. The conventional supports can tolerate pressure only up to a particular limit. the smaller particle size and the enormous pressure. HPLC is therefore highly effi cient and has a very fast speed of resolution. the pressure has to be Increased. Lentil lectin is used to separate histo-compatlbility anti gens. Wheat germ agglutlnin is used to separate dif ferent types of lymphocytes. if one uses a longer column. one can increase the flow rate thereby reducing the retention time of the solute components. There are several kinds of lectins and each lectin has its own specificity. Due to this. The technique is primarily suitable for analyt . Lectins are normally isolated from pla nts and a few other sources. a conventi onal chromatographic experiment takes an inordinately long time. 405 Lectin-sepharose. This method uses an extremely high pressure (up to 8000 psi).e . band broadening is minimized. Normally they recognize a specific sequence of sugars or sugar de rivatives in the given polysaccharideYglycoprotein.ChromatoraplJ 6. To increase the flow rate. it also reduces the effi ciency of the column because the solute components undergo lesser number of equilibration. i. Although this manoeuvre reduces the time of experiment. To reduce the time o f experiment. To circumvent this. There is no loss in efficie ncy also because the supports that are used here are different inasmuch as the support particles are very small and more or less uniform in size. HIGH PERFORMANCE LII/ID CHROM¢OGRAPItN" ". All t hese factors were efficiently resolved with the development of high performance liquid chromatogra phy (HPLC). the diffusio n effects increase and the peak is broadened. "tble 11. None of the other techniques discussed in the previous pages are so v ersatile.15 Experimental Differen¢ BetwRn Coaventional Chromatzogzaphy end HPLC Conventional* Number of plates/second Pressure Flow rate (mm/min) Time of experiment Equipment Purpose Quantity chromatographed 0. It is particularly popular for the separation of polar compounds such as drug metabolites.ical purposes but can be used as a preparative technique also. in general. which. are poorly resolved by other techniques. .15 summarizes the differences between the conventional column chromatography and HPLC. and affinity chromatography. Perhaps the greatest advantage of HPLC Is that tt may employ the pri nciples of adsorption. column and accessories Predomanfly preparative mgto kg 5 Upto 8000 psi 6OO Minutes to hours Integrated chromatograph Predomlnanfly analytical ng to mg *Values for Gel-F11teration have been used. Thi s makes it an extremely versatile technique and explains its emergence as the most popular chr omatographic technique. Table 11. ion-exchange. exclusion. partition.02 Negllgble 5-50 Hours to days Simple. . (/v) A column in which the separation will take place.23 presents a schematic diagram of the instrumentation required for HP LC. (//) High pressure pump to push the mobile phase through the column. Solvent Resesoir and the Solvents The solvent reservoir should meet the following criteria: (0 it must contain volume enough for repetitive analysis. (v) A detector used in detecting the concentration of the sample components as they come out of the column.23 Schematic diagram of an HPLC system Figure 11.406 Column Biophysical Chemistry [ Solvent ervolr microfllter High Pressure pump Sample --. (vi) A potentiometric recorder to produce a chkomatogram.to the mobile phase. Six major components needed to perform HPLC are (/) A solvent reservoir to store the mobile phase.Injection port Guard column Detector Recorder Figure 11. (//0 A device to inject-the sample in. . The solvent container should preferably be insulated aga inst contamination through laboratory atmosphere.0 liter ca pacity are suitable as solvent reservoirs. Solvent degassing witPn the reservoir is performed usually by heating or by appl ication of vacuum or by treating it with ultra sonic sounds. Occasionally sparging with helium might be used for degassing. however. Glass bottles in which the HPLC solvents are sold also ma ke for a very good solvent reservoir.5-2. glass and steel containers of 0. The type of separation desired dictates the choice oRhe mobile separations can be carried out with a single solvent or a fixed proportion lvents. .(//) it must have a provision for degassing the solvents. Isocratic mixture of two so changes continuou assembly. Generally. (///) it must be inert to the solvent. the development solvent composition sly. This is achieved by a gradient programmer usually attached to the HPLC phase. For gradient elution. ('/) A constant volume delivery. all the solvents are degasscd by met hods specified in the preceding paragraph.. (/v) Reciprocating piston. Flow rates are dependent upon column permeability and the gas pressure a pplied. (/v) Amenability to high pressures of up to 6000 psi. and reproducible quantitative analyses. and cannot be used fo . Since air bubbles interfere with continuo us monitoring of the effluent and with resolution itself.good pump should have the following quali ties: (/) A pulseless stable flow. (//) A suitable pump should provide solvent flow-rates of 0. the pump is a major factor in obtaining high resolution.Chromatraphy 407 All solvents to be used in HPLC must be extra pure since even the smallest impur ity interferes with the detection system. (0 Holding coil. By producing reproducible hi gh pressures. These pumps can at best provide pressures up to 1500 psi. A .5 mm filter placed before the pump. A large ' holding coil made up of stainless steel tubing is filled up with the solvent. high speed a nalyses. (v) Syringe pumps. even the extra pure HPLC solvents are passed throu gh a I. Pumping Systems Pumping system can be said to be the heart of HPLC. Thus. Pumping systems available for HPLC are: (/) Liquid displacement by compressed gases (holding coil). Absence of pulsations minimizes detector noise. Compressed gas from a cylinder forces the liquid at constant pressure from the holding coil into the chromatographic column. (v) The pump should be adaptable to gradient operation. which is compatible with most HPLC modes.5-10 mYmin. This is more so if the detection system is measuring the absorbance below 200nm. This unit is usually available with less expensive HPLC systems . (/0 Pneumatic amplifier (///) Piston/diaphragm driven by a moving fluid. r gradient elution separations.24). The pump has a piston driven by the compressed gas (Figure 11. These pumps are consequently not very popular. Another disadvantage of these pumps is that many times the driving gas can inadvertently get dissolved in the mobile phase and cause problems in resolu tions. (i0 Paumakie ampler. This pump also uses compressed gas for pressure. The pump uses gas at c omparatively To column . These pumps give a pulseless flow and they are adaptable to gradi ent elution.i. The . Figure 11.Column Mobile phase Piston Reservoir. The gas is in contact with a large surface area of the piston. The pressure of the gas is thus increased 10-20 fold when it is applied to the solvent. Check valves Figure 11. Hydraulic fluid Flexible diaphragm To column MoWr 408 Biophysical Chemistnj low pressure of about 200 p. This pump gives a pulsel ess flow and is ideal for quantitative purposes. On the backward move the piston sucks in solvent from the reservoir. I||/)/ov/ng fluhi type. A smaller surface area of the piston is in contact with the solvent. These pumps use a piston that is in direct contact wit h the solvent. which it pushes into the column on the forw ard move.s.25 Schematic of a moving fluid type pump Iis Reciprocating piston.25 is schematic for this type. The piston may be driven with motors and gears or by solid-state pulsin g circuits. A piston moves rapidly back and forth in a hydraulic chamber. These pumps use either a piston or a diaphragm driven by moving liquid. outlet to the columns close during the backward move to maintain the pressure in the column. . be filled with the An appropriate valve then channels the eluant fom the pump through the loop dire ctly the column. glass.26. c opper. the sample can be injected while the system is under hig h (/0 The second method employs a small volume metal loop which can . which allows efficient packin g. (v$ Syringe pump. (/) The first method employs a micro syringe designed to withstand high pressure s. With help of this micro syl ge. Preferably the sample is injected when the pressure dropped to almost one atmosphere after switching the pump off. however. The sample is thus carried spontaneously with the eluant to the colu mn.Chromatography 409 The pump. As shown in Figure 11. as for all conventional types of chromatography. Stainless steel columns are preferred since they can withstand pressures up to 8 000 Straight columns of between 20-50 cm in length are generally used. Packings . The pump is well suited to gradient operations. The internal diameter of the columns is usually 14 ram. This technique is known 0jectWn. Two methods are available to introduce the sample as a narrow band. packing material is supported by a porous stainless steel or teflon plug/disc at the end of column. columns usually possess an internal mirror finish. The pump is not very popular. whereas for other longer colunms are necessary. The columns for HPLC are usually made up of stainless steel. an important factor in achieving a satisfactory resolution. these pumps operate . Short uired for liquid adsorbent and liquid-liquid chromatography. aluminium. In order to suppress the p ulses a pulse dampening system has to be employed. Sample Injection Sample introduction on to the HPLC column is. fails to produce a pulseless flow. These pumps provide a stable flow rate high pressure. the sample is introduced either directly onto the col umn or onto directly above the column. Alternatively.by a screw gear displacing a plunger through the solvent reservoir. Coarser particles induce increased band broadening. To (s) 11. .Maximal separation without or with minimal band broadening is a function of the and mobile phases chosen.27 Schemat/c representation of the strcture of three types of supports common ly used for HPLC. g. These supports are superficially porous (Figure 11. However.27(C)). (i//) bonded phases where the stationary phase is chemically bonded to an inert {Figure I 1.27(B)).16. The stationary phase is a compromise between the above factors.410 Biophysical Chemistry minimize this unwelcome phenomenon. Table II.tionary supports for different modes of HPLC have been described in 11. Clearly then the smal l particles should i also be able to withstand the increased pressure that they will be subjected to. a glass bead of about 40 mm diameter). as the particle size decreases the resistance to solvent flow through the column increases and a higher pumping pressure is required to maintain a satisfactory flow rate.27(A)).16 A Lis-of Stationary Phases used in Various odes of HPLC Mode Adsorption Partition Exclusion Ion exchange Nature Commercial name Type of support Applications Silica Alumina Silica . stationary supports for HPLC have been so de signed that the individual particles are as small and as uniform as possible. Individual sta. (//) pe///cu/ar supports consist of a solid inert core onto which are coated sev eral particles (e. Three forms of column packing m aterial are available'based on a rigid solid rather than a gel structure:. These pa rticle are generally 5-10 mm in diameter (Figure 11. (/) m/croporous supports where micropores ramify through the particles. . Bondapack-NI-I Blo-Glas Styragel Sephadex Partisfl-SAX Mlcropak-NH2 Partisfl-SCX Zipak-WAX Pellicular Pellicular Mlcroporous Microporous Pellicular Porous Porous Rigid solid Semi-rigid gel .Alumina Octadecylsilane Octadecylsilane Alkylamine Glass Polystyrene divinylbenzene Agarose Strong base Weak base Strong acid Weak acid Corasll Pellumina Partisll MlcroPak A1 Bondapak Cs Bondapack . nucleotides. The hard gel. chlorinated pesticides. nucleic acids. pesticides. polar herbicides. Care should. however. peptldes. method known as high pressure slurry technique is used for packing the column. vitamins. fatty acids. The packing has to be uniform without any crocks channels for obtaining optimum separation. Dansylated amino acids. These gels. Prepacked columns are also available and can be purchased from the market. The technique can be extended to hard gels. The columr a porous plug at the bottom. Column Packing Procedure. proteins. have to be into the column under gravity in a way similar to that of conventional chromatog raphy. The slurry is now pumped into the column at high column so packed is then equilibrated for a long time by passing the developing solvent it. nucleot/des. Rigid solids and hard gels should be packed densely as possible. however. The Guard Column The resolution power of HPLC is so high that an elaborate sample preparation . Proteins. this technique cannot be because pressure results in the fracture of gel particles. Amino acids. A suspension of the packing material is prepared in a suitable solvent. however. For soft gel. polysaccharides. drugs. therefore . be taken to not to fracture the parti cles. polysacchar/des. triglycerides.Soft gel Porous Porous Porous Pellicular Steroids. has to be swollen before it is pumped under pressure. peptides. aflatoxins. or other biological materials can b e applied .chromatography is not necessary. Thus. sera. Since the quantiW of the sample applied to the analytical column is small.411 This. . fatty acids. Apart from the above two. These can be either recording spectrophotometers or manual wavelength spectrophotomcters. clogs the column after a few applications during separation retains many undesirable components of the biological samples.. a short column (2-10 cm} precedes the main column. are however. The guard column has they same diameter an d same packing as the main column. aflLnlty. are by these detectors. it is important discuss how the choice of a mode is made for a given sample.).. as only those sample molecules which absorb at 254 nm or nm can be detected.28 ttlust rates the Affinity] F Non-ionic Reverse . Thus. and well suited for gradient elution. refractive index monitors and fluorescence detectors a re also with HPLC systems. This short is known as guard cownn and its function is to retain those biological component s would otherwise clog the matn column. ion-exchange etc. Flgure 11. UVNIS photometers can be used for HPLC.g. however. sample selective. The packing of the guard colunm can be replaced at intervals. hydrocarbons etc. it is imperative the sensitivity of the detector is sufficiently hlgh and stable. The advantage with these is that a range of biological subst ances detected by selection of appropriate wavelengths. normal flow and temperature fluctuations. of Separation System Since HPLC can be used in many modes (e. Crcumvent this problem. sensiti ve. carbohydrates. liplds. These detectors are inexpensive. UVNIS spectrophotometers with wavelength selection range of 200-800 nm are very popular detectors. 28 Gulde W HPLC. functlonal. Compounds possessing different functional groups are ideally . Organic in molecular size Molecular solvent "[ exclusion Different J Homologous Normal Adsorptlon ------. Biological Aaueous in molecular size I Molecular specificity /. mode selectlon It is usual to separate compoUtlds soluble in organic solvents by either partiti on or chromatography.lvent ] exclusion Large derences. series phase go ups Flgure11.low polarity phase / Large differences. 412 Biophysical Chemlstr separated by adsoHon comatoaphy on sca a non-pol solvent as e ioc or non-ioc substces which e water soluble eld to sepation phase pHon mode. However.The reverse phase system is therefore very useful for separation of non-po lar solutes. e oy difference being at e colums is of a lger term dieter (10-20 ) so that a 1ger volume of e sple c be processed. Specialized Techniques: Reverse Phase Chromatography As opposed to the usual polar stationary phase and a less polar or non-polar mob ile phase. their retention times will also increase since they will interact more with the non-po lar stationary phase. an extremely polar solvent becomes the weakest eluent here. For is prepative HP may be peffoed. Wey by rerse should be sepated applied epative If the study involves chacteaon d fuher studies on e sepated component. Repeated appleton of e sple d coec0n of effluent fractions contng e desired components c we a f eld ofe component d interest. Therefore the driving force for retention of a component is not its interaction with the stationary phase but the effect of the mobile phase in forcing the component onto the hydrocarbon bonded stationary phase. epaflve HPLC c be ced out using these pacing mater. polar substances will interact more with the polar mobile phase and elute first. As the non-polarity of the solute components incre ases. fever phase ption mode c so be to these subsces. Therefore homo . Moreover. Methanol and acetonitrfle are stronger eluents than water. On e oer hd songly ioc solutes soluble water by e ion-exchge mode.s d solvents. the stationary phase in reverse phase chromatography is hydrophobic (hydr ophobic bonded phase usually possessing Cs or Cs functional groups) and the mobile phase is pohar (fully or partially aqueous). it is perave that a lger qu of the pure components is needed. mobile phase. In this case. Solvents of intermediate eluting strength can b e obtained by mixing one of these solvents with water. Water. We have already seen while discussing the nature of partition forces that non-po lar substances are squeezed out of the polar phase. th i$ 'squeezing out' is proportional to the non-polar surface area of the solute. is techque is o as on-paa (see later). When the sample has many components covering a wide range of polarity. In this mode the stationary phase is non-polar (hy drocarbon) and the mobile phase is a polar (water/acetonitrile. a gradient elution has to be carried out for optimum r esolution. Thus by adjusti ng the pH we can alternately suppress the ionization of acids and bases and other dissociable so!utes and make them separable by reverse phase technique. The buffer concentration in such experiments is usually maintained at a high level. especially when ionogenic substances (dissociable solutes such as acids and bases) are to be resolved. In many applications. These gradients are prepared by continuously decreasing the polarity of the eluting so lvent. bases will rema in unionized and therefore will yield themselves to reverse phase resolution. the eluent pH must be controlled by using a buffer. If the p H is adjusted on the acid side. Adjusting the pH on the basic side. water/methanol) mixture. Ion suppression. For separating weakly ionic substances also. ionization of acids will be suppressed. This can be achieved by gradually increasing the content of organic solvents (methanol or acetonitrile) in water/organic solvent mixture. This will allow the acids to be separated by the reverse phase technique. reverse phase chromatography Is preferable. This approach is termed ion supp ression and Is useful for resolving weak'acids and bases within the pH range 2-8. This allows protonic equilibria to be .logs show a good resolution by reverse phase chromatography. 29 illustrates the pl'inciple ofion-palring. Sinc e phases are not stablein high alkaline pHs. At low pH. However. Strong acids and bases do not yield to resolution by ion suppression sensitivity of siliceous bonded phases to extreme pH values. This counterion forms a ion pair complex with the sample long. The e. By adjusting the pH in a manner sample components are present in their ionic forms and by choosing a strongly io nic group attached. (to its concentration. The complex now behaves as a neutral. This can be achieved by fonnlng an ion pair (a coulombic asso ciation arising when two long bearing opposite charges associate) with a suitable counte rion. use of alkaline solvents is precluded . The ion pair formation and consequently the resolution of sample is affected by (/) the size of the counterion. Counterion+ }o pair Flgure 11.A-tent to which the ionized sample and counterion form an ion pair the retention is increased. the situation can be expressed by the following Solute" + Counterion* = (Solute+. can also be resolved by reverse phase technique ff they can be converted to ' neutral species. lon.413 rapidl"and therefore does not allow asymmetric peaks and band splitting due to t he of secondary equilibria involved in the chromatographic process.pa|ri. and (rio t he pH of solution. The commonly used counter ions include I chain aliphatic amines for negatively charged sample long and perchloric acid for positively sample long. perchloric acid is usually the choice. Phosphate buffe r is for moderate to high pHs. {B} POLAR PHASE (MOBILE) {HA) Weak Weak base acid . trick is to add a large organic counterion to the mobile phase. Ion pair ..lipophi lic lipophillc ion base NON-POLAR PHASE {STATIONARY) Figure 11. (A) aq Conjugate + Counter Ion pair .29 Diagmmmatlc representation of prlnciples of ion-pairlng . Counter.(BH÷1 aq (F-) aq {HBF} {AF) (l) aq-. + Conjugate' acid ion . H + Cl" Suppressor oolumn Resin-N+ O1'+ H +Cl'---'Resln-N + C" + H20 Resln'N÷OH-+ Y+CI'Resin-N÷CI" + Y+OH" Conductivity cell Eluent : 0. Ion chromatography solves this problem of detect ion by using an additional suppressor column situated downstream of the separator column. anion-exchange column is followed by a cation-exchange suppressor column.. NH. Although strongly ionic substances can be separated by ion. These limitations are usually due the limitations of the detectors employed. This s uppressor column is so manipulated that it neutralizes the undesired long allowing to pass through the detector.SetH ++ Y + Cl'q==Resin-SOY +. The su ppressox column then removes the long of the eluting base: CATION ANALYSIS Y+-m No+. Thus. there are ]imitations to this process. Unless the ion coming out of the colu mn has directly measurable property such as radioactivtyr UV absorption. for an ion analysis. which is usually a conductance cell. continuous mo the effluent becomes difficult. tthylsmlne trlethylsmine Resin.Separator column WoMe ample Eiuent Injection 414 Biophysical Chemistry Ion chromatogrcphg.exchange mode.01 N HCI . oppmethylmlne Min : rio ppm trlethylamine .Flow: 0 5 10 15 20 25 :. 3 ppm SOt.4 ppm Fb. ©. 8. The eluent u .9 ppm NO d.Na÷ + H20 e anions to be separated get converted to their respective acid forms: resin-H÷+Y÷+A.6 ppm NOs.resin . 14. . ppm Cl. Flow : 138 ml/hr DMector : 10p mholcm full scale Peak Identity : t a.003 M NaHCO3. 143 ppm P043" Min f. 2.H÷ + Na* + OH. g. 1.-> Resin.30 Flow scheme of ion chromatography (reproduced from Dione.resin-Y÷+H÷+Athrough the monitor where they are detected by the conductance cell.For cation situation will be reversed and the separator column used will have a cation exch anger whtle the suppressor column used will have an anion-exchange resin.SONa÷ + H÷X" 1 -Waste Fluent :0. resin .415 Resin-N+ HCO + NaX'Resin-N+X" + NII+HCO 1 Reeln-80H÷ + NHCC--Resln-SONa÷ + FICCj Resin-803"H÷ + Na÷X'. Corporatio n). Fjure 11. sed HCI. . 416 Biophysical Cherntstr The separator columns ususlly employ special low capacity resins while t he columns employ conventional resins with high degree of cross linking. NaHCO . The choice of eluent is a compromise between eluting power and The choice of the eluent is dependent on the relative affinities of the eluent i on and the long for the resin. Na÷. nucleic acids polysaccharides. Ca=÷. HCI. Routinely separated long include F-. CI-. NO . Applications of HPLC In recent times HPLC has emerged as a method of choice for analytical purposes. f or phosphoproteins. and NaOH are most commonly used eluents. CrO -. and NH etc. biggest advantage that it. the binding is an attr/bute of these phosphate groups. Mg=÷. drugs and animal and plant hormones and complex lipids. Arttflclaliy it may be prepared from CaI-IPO4. SO -.Ca(OH)2]. The sample requiremen t is very low for this technique and as less as a few femtograms of the sample will b e satisfactorily. and chromate. amino acids. for GLC. HPLC has been successfully applied to the separation of proteins. steroids. On top of all this the detectors that are employed in HPLC nature and thus the separated components can be recovered for further study. plant pigments. AgNOz. is times more than other techniques except. SOME SPECIALIZED TECHNIQUES Hydroxyapatite Chromatography Hydroxyapatlte is a crystalline bone mineral. Thus. pesticides. There are several other theor/es about the binding and we kow that electrostatic effect is not the only factor involved. perhaps. It is assumed that atleast for those wh/ch contain phosphate. RNA. has over other techniques is the speed of analysis wh ich. compomds bind at a low phosphate concentration and c be eluted out by increas/r concentration in the eluting solvent. DNA. Both the ion species should have roughly equal affinities fo r separation.2H20 crysta ls. PO-. Columnsc hydroxyapat/te interact and bind those substances which interact with calcium. Its composition may he given {Caz(PO4)213. Hydroxyapatite chromatography is extremely usefulin nuclelc acid esearch where i t . Since melting temperatu re for DNA r/ch in A. The concentration of phosphat e in the eluant is now ra/sed sl/ghtly only to a level wch is sufficient to desorb single -stranded DNA.and double-stranded DNA is appl/ed to a hydroxyapat/te c olumn. these DNA molecules wfl/be denatured and converte d to single- . DNA from different sources dLfers in the base composition. Now the temperature of the column is increased steadily. If a mixture containing both single.T base p airs is known to melt (conversion to single strands by denaturation) at a lower temperature as compared to the double stranded DNA r/ch in G.C base pairs. A sl/ght increase in phosphate concentration o f the result in selective desorption of the single stranded DNA. both forms will in/tially be adsorbed. i./ons can be used f or fracUonating DNA according to base composition. with respect to its contents of A. The double-stranded D NA can now l separately eluted with further increase in phosphate concentration of the eluant .T and G. Hydroxyapatite chromatography coupled with temperature var/at. Double stranded DNA rch in A.e. This procedure is known as therm al chromatography.C base pa/rs.T base pairs is low.. Im.e a system where DNA from var /ous sources d/ffering in their base compositions has been appl/ed to a hydroxyapatit e colurnn: Al/ these double-stranded DNA will bind to the column.ch/ef use is in the separation of single-stranded DNA from double-stranded DNA. Th e l)olyacrylamide is made so that it has a large pore size to minimize molecular s ieving. thermal chromatography can achieve fractionati0n to the base composition. use of hydroxyapatite chromatography in protein research is to Concentrate lrote ins a very dilute mixture. all DNA fragments bind tightly. As the salt concentration is increased. The perchiorate can then be removed by or by passage through a desalting gel. Affinity System for Fractionating Supercoiled and Non-Supercoiled DNA This affinity system consists of the dye phenyl neutral red immobilized onto gel of large pore size. Hydroxyapatite columns are useful for separation of phosphoproteins also. An Affinity Bystem for Base Composition Dependent Fractionation DNA A polysaccharide gel bound to the dye malachite green is used. At low salt concentration both supercoiled a nd coiled DNA bind to the column.givs a good esolution. Howeve r.Chromatography 417 molecules first and wi/l elute out at a low phosphate concentratlon Tile G.C pairs. the nb . This dye binds more tightly to single-stranded regions o f is an attribute of supercoiled DNA.mechanism of such binding is not Nevertheless. Needless to say that the. this method is very useful in protein separation and many times wh en no gives the desired result. Elut ion is now carried out by increasing concentration of sodium perchiorate (NaCIO4) in the eluant.T content elute out at a lower perchiorate con centration as to those fragments which are rich in A. Only when the temperature olumn has risen sufficiently high to denature DNA molecules. DNA fragments which are poor in A. If DNA fragments (most of the DNA fragments generated by restriction digestion) are loa ded onto a polyacrylamide immobilized malachite green.T pairs. hydroxyapatite chromatography. Thus. what is that even those protein molecules which possess no phosphate groups can on such columns. Malachite green h as a of binding strongly to A.T pairs as compared to its binding to the G. will these molecule s elute out. This sequential elution effectiv ely DNA on the basis of base composition.C rlc h DNA molecule still remains adsorbed to the column. A higher salt concentration is required to el ute super coiled DNA. this results in . Thus after binding DNA mixture to th e column.auper coiled DNA elutes out first. The low ioni c of the buffer helps in such and adsorption. The dried powd er is to remove unboundDNA.or double-stranded) and drying the mixture under vacuum. This has made this affinity system very popular for fractionating these two The phenyl neutral red column is also used to separate DNA on the basis of base As opposed to the property of malachite green binding preferentiallyto A. the DNA-binding proteins adsorb to the immobilized DNA. the dried light. As the mixture passes the column. dye binds preferentially to G. Chromatography DNA-cellulose columns are used to separate DNA-binding proteins from non-binding Preparation of DNA-cellulose consists of mixing cellulose powder with a solution of (single.C rich DNA fragments can be eluted out sequentially by increasing the salt the eluant. The procedure chemically the two substances. A mixture of diffe rent proteins a low ionic strength buffer is layered on a DNA-cellulose column. The procedure of DNA-ceIlulose chrornatography is as follows. poor and G. Washing of the column at this stage elutes The ionic strength of the eluant in now gradually raised. To couple the DNA more tenaciously to cellulose.T pair.C base pairs. compounds contain at least one asymmetric carbon atom or are molecularly asymmet ric. These isomers differ in physical properties even though they have the same functional groups. it is the L-isomers of amino acids which on e finds in proteins and the D-isomer is biologically rejected. These stereoisomers can be separated from one another using the conventional chromatog raphic techniques. which allow mixtures of enantiomers to be resolved. Chiral Chromatography Many of the biological and pharmaceutical compounds are chiral in nature.418 BWphys. Till sometime back this was impossible. Chromatographic techniques. Diastereoisomers are optical isomers without an object image relationship. It is the D-glucose. Thes have identical physical and chemical properties and differ only in their in their optical properties.al Chemtr desorptioIi of DNA-binding proteins in a manner that the strongly binding protei ns elute last at higher ionic strengths. are collectively called chiral chromatography. It is usual that Just one isomeric/enantiomerlc is biologically active. But the advent of chlral chromatography has made it possible to a certain degree. not L-glucose th at has been preferred by nfe. Thus (D + L) + D DD + LD . repressors. This method has become the byword for purificati on proteins as the nueleases. The diastereoisomer approach means that enantiomers contain a functional group t hat can be derivatised using a chemically and optically pure chlral derivatising age nt (CDA) so that they become d mixture of diastereoisomers. For example. It is thus a challenge to the pharmacists as well as biologlsts to separate the racemi mixtures of the two isomers/enantiomers so that the biologically active amongst them may be obtained in pure form. polymerases and many other enzymes and which bind to DNA. T hey will eventually allow racemic mixture to be resolved. albumin. Chiral mobile phase agents that have given good results are 10camphorsulphonic acid. One form of the stationary phases used here is proteins (bovine serum albumin) since proteins themselves are optically active. Perhaps the most successful method for chiral chromatography has been to use a c hiral stationary phase. Due to the different spatial arrangement of the functional groups at the chiral center of the enantio mers.Mixture of Chlral derivatising Mixture of enantiomers agent diastereorners Note that the two diastereomers will have different physical properties and can now be separated by conventional chromatography. This approach is based on the basic premise that there would b e a threepoint interaction between the stationary phase and the stereoisomers. A variant of the above technique is to use a mobile phase that itself is chiral. These transient complexes may usually be f ormed by hydrogen bonding and wander waals forces. . In spite of the basic simplicity of the above approach it has suffered on accoun t of its being slow and not really quantitative. This leads to the formation of a transient diastereoisomeric complex between the enantiomers a nd the chiral mobile phase agent. and N-benzoxycarbonyglycyl-L-proline. It is usua l to prefer reversed phase chromatography for chiral chromatographic separations. different transient complexes of stationary chiral phase with the enantlomers will form. Alternatively. Pyridine-2thione absorbs at 343 nm and therefore the binding of a protein to the matrix ca n be spectrometrically followed. This is achieved by t reating the matrix with 2. Alternatively. The covalenfly bound protein is now displaced by using a solution of a mucl high er concentration of dithiothreitol (50 nM).dipyridyldisulphide.containing protein will interact with an immobilized ligand containing a disulphide group. pyridine-2-thione is formed and the protein becomes covalenfly attached through formation of a disulphide bridge . The procedure which can be classified under affinity chromatographies takes advantage of the fact that a thlol (--SH). The scheme given below illustrates the chr omatographic procedure. The last step is to regenerate the ligand for further use. cysteine or glutathione may also be used. The column is now washed to remove non-thiol protein s.Matrix Matrix Matrix Matrix Chromatography 419 Covalent Chromatography This chromatography procedure was speclflcally designed to fractionate thiol-con taining proteins. When a thiol containing protein interacts. one may also use mercaptoethanol for this step.2'-. The ligand : 2'-pyrldine poSH thlol protein . A weak solution of dithiothreitol is used subsequently to remove unreacted thiopyridyl groups. The most used ligand in this chromatography is disulphide 2-pyridyl group attach ed to an agarose matrix. --/ S--S-. 2'-Dlpyridyl disulph/de The ligand N SH H + 2-Thiopyridine Thlone .P Protein attached to the matrix Add dlthlthreltl Puttied protein 2. ed. . J. 5. 1977.420 Craig. Physicochemical Applications of Gas Chrom atography. Vol. Amsterdam. Equipment Techniques. Van Nostrand. Vol.. S. 7.}. 1977. R. and Horvath. lon Exchange. London. K. 1972. 6. Lederer.. Grant. Chromatography. and Pecsok.. L. The classi c paper which described partition chromatography. 35:91 {1941). Analytical lon Exchange Procedures in Chemistry and Biology --Theory. New York. K. Wiley Interscience. R. 3: Liquid Column Chromatography (Z. 1979. M. Wiley Interscience. 2"d ed.. G. Foundations of Modem Liquid Chromatograph y. McGraw-Hill. Ettre. J. pp.. J.. Helfferich. 1975.S. C. 2. L. 1978. part I.. New York. 1 4 (Perry... F... R. Kirchner. 47:422 A. Journal of Chromatograph. Modern Reading I. L. J. R. A.. 1975.. Elsevier. Biocherru J. New York. Elsevier. Grob. 1971. 2"d ed. R. and Craig.... Martin. Weissberger. and Kirkland J. Vol. Khym. Gas-Liquid Chromatography. Modem Practice of Gas Chromatography. Laub. 3. N. Prentice-Hail. Library. 1974. Macek and J. R. 8.Chert. 4.J. 195 7. Snyder.M. L. eds. E. "ed.P.). L. New York. and Synge. Janak. 9. Engelwood Cliffs. in Techniques of Organic Chemistry {A. Interscience. New York. Wiley Interscience. Anal. D. L. Introduction to Modern Liquid Chromatogr aphy. C. 2.. L. 1956. 3. New York. Thin-layer Chromatography in Techniques of Chemistry. Deyl. New York. and Lederer. Ill. Wiley.L. 52-392.). J. M. M.) 3 edition. Pece... 6 ed. Anal. Acad. and Ashworth. J. C.97).. Afftnlty Techniques. Wilcheck. 15. B. Academic Press. P.. Sci. H. L. N.) Vol. R. eds.. 61:636-643. Willare. Elution was started and the f ractions Biophysical Chemistry Suggestions for Further Reading Classics . 14. Karger. and Settle E. Cuatrecassas. Affinity Chromatgraphy Wiley. Jr.98). Freifelder. Chem. L. R. Engelwood Cliffs. Freeman. A solution containing glutamlc acid (pl = 3. B. 50:1048 A. J. Prentice-Hail.. 16. W. John Wiley and Sons. 17. L. A. 1978. 1978. New York. The original pap er on affinity chromatography.. and histidine [pl -. Jacoby. vallne {pl -. H. 529-564 (a good text on HPLC) CBS. B.. 1982: 12. A. Proc.. alanine (pl = 6.. 1974. le ucine (pl = 5. D. Selective Enzyme Pur ification by Affinity Chromatography. Natl. Wflchec k. Jr. Experimental Techniques in Biochemistry.7.10. and Dean P.. Reversed Phase Liquid Chromatography and its Applications to Biochemistry. Physical Biochemistry: Applications to Biochemistry and Molecular Biology. New York.22). 11. A. May. G. 13. Lowe. Brewer. and Anfinsen. J. Affinity Chromatography in Separation and Purification {Perr y and Weissberger. eds. H. 1986. pp. W.02). Ins trumental Methods of Analysis.. W. San Francisco. New York. New Delhi. B. C.. R. D.. 1974. 1968. Dean. Merritt.5. xercise I. and Giese.. USA. Methods in Enzymology (W.0 citrate buffer was applied to a cation exchange column that was equilibrated with the same buffer.58) in a pH 3. 34. S. 1974. and M. .1. Can you use ion exchange chromatography for their sepa ration? 5.000 D.000 D. B. B has a molecular weight o f 91. Three pure preparations of different proteins are mixed together and the mixture is run on a gel filtration column. A given solution contains three proteins A. Protein A is unstable above its isolonic point while prote in C is unstable below its isoionic point. Give reasons for your answer. can you choose for its alilnity chromatographic separation? . State the order in which you think the five amino acids will elute fr om the column. Protein A has a molecular weight of 62. 2. What are your conclusions? 3.2 and 7. other than a competitive inhibitor.1. The pIof the three proteins are 5. Wt. only two peaks are seen during elution. Rather than getting three peaks as one would expect. It is normal to select a competitive inhibitor as ligand for affinity ch romatographic separation of an enzyme. You have a protein preparation which contains two enzymes of similar mol ecular weights. what ligand.6 respectively. 4. If the enzyme carries out a single substrate reaction requiring the pres ence of a metal ion. 6. By activity studies you determine that while one enzyme retains its activity at a temperature of 41°C. 6.lotein Mol. the other seems to have become denatured at the same temperature. Devise a procedure for their complete solution. Devise a chromatographic procedure for the complete separation of these two enzymes. Suppose your objective is to separate an enzyme that carries out a bisubstrate reaction. Will it still be necessary for you to select an inhibitor as ligand? 7. 421 collected. Give reasons. Protein C has 11 cysteine residues while the t wo proteins lack cysteine.500 D and protein C has weight of 90. and C. 000 Daltons. I used a much larger amount o f the source than I usually do with a view to increase the yield of finally purified e nzyme. Following is the elution volume data obtained from experiment Elution volume (ml) Myogiobin Serum Albumin Catalase Unknown Protein 6900 68500 221600 ? 118 58 24 37 What is the molecular weight of the unknown protein? 10. A few days back I thought that I can use my expertis e in preparing this enzyme commercially and selling it.000 to 4.00. What may have gone wrong? 11. serum albumin. In this column myglobin. catalase and an unknow n protein were migrated. Elution is now started by lowering the pH grad ually. Malate dehydrogenase carries out a reversible bisubstrate reaction using NADH as one of the . I have been purig/ing it from a given source by gel filtration. Which of the tw6 peptides will elute first? . The results are al ways good and have been standardized. substrates. A tetrapeptide and a heptapeptide ofglutamic acid have been bound to an anion exchange column by using a buffer of pH 6. I work with a particular enzyme during my research.8. Rather than getting that. Can you use NADH as a ligand for the separation of the enzyme by a ffinity chromatography? 9. I found that the enzyme activity was eluting very quic kly. Rather than buying t his enzyme. A molecular exclusion column is designed with a protein fractionation ra nge of 5. . almost all particles (e. Arrow 2 indUrates faste r movement of a particle bearing a net charge density of-2. Although Reuss had observed the flow of water through clay (electro-osmosis) and the migration of clay in the opposite direction due to an electric current. although depe ndent uPon the Figure 12.) and many important biomolecules (e. and Maclnnes. however. peptides. was instrumental in popularizing. bacteria etc. These groups determine the net charge density of the protein molecule which makes it move in an electric field in a direction and at a velocity dependent upon the sign and quantity of this net charge density (Figure 12. The usual purposes for carrying out elec trophoretic experiments are () to determine the number. and (tO to obtain information about the electrical d ouble layers surrounding the particles.12 ELECTROPHORESIS Electrophoresis is the migration of charged particles or molecules in a medium u nder the influence of an applied electric field. nucleic acids. Example may be cited of protein molecules.g. Huckel. Debye. Th e most important early theoretical studies in electro-kinetics were made by Helmholtz. nucleotides..g. the firs t recorded measurements of electrophoretic phenomenon were performed in 1861 by Quincke. which have a large number of ionizable amino and carboxyl gro ups on their surfaces. This net charge density. and mobility of components i n a given sample or to separate them. I A charged particle rngrates towards the oppositely charged electrod e. methods employing stabilized media have become very u seful to the biochemist and will be discussed in the present chapter. Smoluchowski. In modem days.1). Upon suspension in an aqueous solvent. amino a cids. The modem day scientists. and DNA sequencing on the other.) acquire either positive or negative charges. . the utility of electrophore sis to the biochemist. The acquisition of s uch charges depends upon the nature of the particle/molecule and the solvent. Abramson.. red blood cel ls. Gouy. Arrow 3 indicates the dtrectlon of f rlctWnal force. amount. Arrow 1 tnd:Icates move ment of a particle bearing a net charged derts{tj of -1. Tlselius. by describing his moving boundary apparatus in 1937. use it for purpose s as diverse as determination of molecular weight of proteins on one hand. proteins etc. . if the acidity of the solvent is increased (H÷ long} the molecule w ill tend to become more positive and vice versa. as descri bed Stoke's equation.electric field. is the of the solution.Electrophoresis 423 groups and their number present in the molecule. Thus F=6rv the friction exerted on the spherical molecule. MIGRATION OF AN ION IN AN ELECTRIC FIELD Consider a situation where a spherical molecule of net charge q Is placed in an electric field. Evenif two molec ules have the same charge.-will depend upon (0 the size and shape of the molecule. they will have different charge]mass ratio (this differences is of more use in electrophoresis on gels). is modified by the nature of th e solvent. they might not migrate together because if there is difference in their molecular weights. Even typically uncharged biomolecules such as carbohydrates can be made to wear weak charges through derivatization as. Taken together. Since the particle has been suspended in a solution.1). and (/ /) on the viscosity of the medium through which the molecule will migrate. r is the radius of this molecule . The extent of the friction. The force Fwhich will act upon this particle will depend upon (f) the net charge density of molecule. This frictional oppose the accelerating force generated by the electric field (Figure 12. Thus. to relationship of electrophoretic migration. we friction occurring between the accelerating molecule and the solution. these differences are sufficient to en sure differential migration when the long in solution are subjected to an . The above rel ationship may be mathematically described as where A E/d is the field strength applied (AEis the potential difference between the two electrodes. and v is the velocity at which the moleculeis migratin g. This is the basis of ¢lectrophoresis. the distance between them). Equ . Electrophoresis in not llmited to charged molecule s only. and (f0 the strength of the field in which it is placed. borates and sometimes as phosphates. (/0 viscosity of the solution (). . The mass consists of ' the size (molecular weight} but also the shape of the molecule.ating force of acceleration with Stoke's equation. above relationship we get the expression 6rld thus be seen that the velocity {v} of the molecule is proportional to (/} the fi eld strength .and (/0 charge (q) on the molecule but is inversely proportional to (/) the par ticle size (r). FACTOR8 AFFECTING ELECTROPHORETIC MOBILITY Saznple Charge/mass ratio of the sample dictates its electrophoretic mobility. we get --q=6nrv d rearrange the. however. More charge is carried per second to the electrode. As an example consider the case of globular and fibrous prote ins. is dependent on pH of the medium. (///) Shape. The importance of resistance becomes clear if one con siders the following situation. This results in a d ecrease in the viscosity of the solution (see Chapter 5). the current increases while the resistance decreases. Rounded contours elicit lesser frictional and electrostatic retar dation compared to sharp contours. Consequently. only a small proportion bei ng carried by the sample long.. The rate of migration under unit potential gradient is referred to as mob///ty of the/on. The Electric Field We have already seen that the force acting upon an ion of charge q is E q/d. Consequently the long present can mov e faster. During any electrophoretic run. One might think that this rise in current would also make the separat ion of sample . Given the same size (molecularreight) the globular protein will migrate faster t han the fibrous protein. greater is the electrophoretic mobility. An increase in potential gradient increases the rate of migration.424 Biophysical Chemistry (i) Charge. The current (total charge carried per second to the electrode) in the solution p laced between two electrodes is carried mainly by the buffer long. greater are the frictional and electrosta tic forces exerted upon it by the medium of suspension. Resistance to current flow is an important point to bear in mind while performin g electrophoresis. The higher the charge. An increase in the potential'difference therefore increases the cur rent. The bigger the molecule. The temperature within the medium rises. (//) S/ze. Le. some power dissipates with consequent generation of heat. Resistance shares an inversely proportional relationship with t he rate of migration and the current. larger particles have a small er electrophoretic mobility compared to the smaller particles. The charge. = solute long . as the temperature rises. .2 If current is not controlled. Increas e in temperature also leads to a decrease in solvent volume and therefore increases buffer concentration. some of th e solvent evaporates increasing the buffer ion-concentration. the temperature within the electrophor etic cell rises leading to decrease in viscosity of the solvent. Temperature . Due to this the buffer ion mobility increases. Consequently the buffer long move faster. = Buffer long . This results in more charge being carried (A) Initial situation l Ewporon i /.components faster. = Evaporation (All solute long are negatively charged and the movement is toward anode) Figure 12. 0./.i/. The sample long move slowly and their separation is slowed down. Th is leads to more curren being carried by the buffer long. Because. This is not so.-----# . However. consequently the buffer long mov e faster. or a cooling system installed (see section on high voltage elect rophoresis). the heat generated is small an d dissipates easily. the whole apparatus mi ght be shifted to a cold room. with cellulose acetate. If the voltage used is high. heat dissipation is not easy and one should therefore maintain a constant current. (0 rpt/on. Adsorption. Electrophorests 425 by the buffer long rather than the sample long. agar.. one should maintain a constant current. Howev er. starch an . But even this inert me dium can exert adsorption and/or molecular sieving effects on the particle thereby in fluencing its of migration. The evaporation of the solvent due to heat generation might be minimized by enclosin g the apparatus in an airtight cover. the applied electric fie ld adsorption. because. Supporting media such as polyacrylamide. power packs have been made available which can control either the voltage or the current to a constant level despite unavoidable changes in resist ance due to temperature fluctuation. which may also i nfluence of sample migration. Ideally. One may therefore use either a constant voltage or current in these case s. means retention of a component on the surface of medium. Thus the rate and resolution of the electrophoretic separation be effectively reduced by adsorption. thus. The component is. even with a constant v oltage heat will be generated and all the events described above will take place. here. not resolved as a sharp band but as a band w hich a tail. and gel electrophoresis. The medium may also give rise to electro-osmosis. Fhe Medium An inert supporting medium is chosen for electrophoresis. rather like a comet. while ithe sample long move slowly and the separation slows down (see Figure 12. Such a component has two forces acting upon it.2). it has been seen that with low voltage paper electrophoresis. (iiJ Molecular sieving. Si nce this should not be allowed to happen. and barbitone etc.I 112. and agarose. citrate. starch. On the other hand. 1966) when electrophoresced pure albumin in the presence of a borate containing buffer. The smaller molecules here pass through the pores easily. He averred that the multiple bands the result of an interaction between borate and some of the albumin molecules]. which can then be separated by electrophoresis. the larger molecules als o are to squeeze through the pores. The electrophoretic mobility is thus modified by molecular effects of the supporting medium (for a detailed discussion see molecular sievin g under Buffer Apart from maintaining the pH of the supporting medium. Borate buffer can. . EDTA. 5:1108. Rat her than one band. Examples may be given of carbohydrates. (i) Composition. In case of polyacrylamide. the buffer can affect th e mobility of the sample in various other ways. phosphate. Care be taken to avoid such buffers. such buffers can be deliberat ely for certain separations.d have cross-linked structures giving rise to pores within the gel beads. B/ochem/stry. however. He extracted the protein from one of the isolated ba nds Again two bands were observed. [A case in point is the example cited below. An artifac t due to ¢ Cann (Cann. larger molecules are retarded.. interact w ith to give charged complexes. Commonly used buffers are formate. In Sepha dex. than the pores are excluded from entering the gel beads and these molecules faster. ac etate "Iris. The choice of buffer depends upon the type of sample b eing buffer can affect electrophoretic mobility if it is able to bind to component(s) sample being separated. he observed -two. J.R. which are uncha rged therefore inseparable by electrophoresis. (lgure 12. (ll/) l/f. As we have already seen above. which has both acidic and basic properties. For an amp holyte such as an amino acid. on the other hand. diffusi on (especially of smaller molecules) tends to be high with concomitant loss of resolution.3) Z H-----OOH -.-O0® Migrations. both the above effect s apply.1M. Since pH determines the degree of ionization of organic compounds. A deci-ease in ionic strength. increased ionic strength of the buffer means a larges share of the current being carried by the buffer long and a meage r proportion carried by the sample long. This situation gets translated into a slower migrati on of the sample components. less heat will be produced. The ionic s trength used is usually between 0. Basic pH Molecule is an anion . it can also affect the rate of migration of these compounds. However.. therefore. Since the overa ll current will be low. would mean a larger share of t he current being carried by the sample long leading to a faster separation.Migration NHs R Migration Migration.426 Biophysical Chemistry (ii) Ionic strength. Increase in pH acids and a decrease in pH increases the ionization of organic bases. Since the overall current will also increase there will be heat prod uction (we have already seen that heat production is detrimental to proper electrophoresis). a compromise. in low ionic strength buffers. H. The chosen ionic strength of the buffer is.05-0. Will they h ave the same mobility? Discuss. * On the basis of the principles discussed so far can you answer the follo wing qu.). TYPES OF ELE . FREE EEC'ROPHORF. They are therefore discussed below very briefly.Acidic pH Isolo.Cs.CTROPHORESIS Electrophoresis can be divided into two main techniques: free electrophoresis or electrophoresls without stabilizing media and zone electrophoresls or electropho resis in stabilizing media. Both the techniques have now become obsolete and are at best of historical significance. 1.estions? (a) Why should electrophoresls be done in solutions of low salt concentration? (b) Two proteins have the same molecular weight and the same charge.3 Dfgram llvtratfng the effect of pH on the fonfzatn and migration of a n amfno acfd. The direction and also the extent of migration of ampholytes hre thus pH depende nt and buffers ranging from pH i to 11 can be used to produce the required separation ( also see Chapter 1). Th e suspension is . Mlcroelectrophoresis -This electrophoretic technique involves the observation of motion of small part icles in an electric field with a microscope (such as IB. neutrophils.IS Free electrophoresis has two main techniques: m/croe/ectrophoresls and mov/ng bo undary electrophoress.nic point Molecule is a cation Molecule is a zwitterion ljure 12. bacteria etc. The ocular micrometer serves for the measurement of t he in conjunction with a stopwatch.Cs.4 Abramson'sJ/at micr0e/ec-rophoretlc ce//. F@ure 12.b. In modern this technique is applied only for measuring the zeta potentials of cells such a s IB. bacteria etc.) This is the prototype of all modem methods of electrophoresis and was first deve loped by Tlselius of Sweden in the 1930s.4.e. -.z x + y + z char at the pH of th b.y. In principle any microscope with a graduated fine focusing tdJustment and an ocular micrometer may be used in conjunction with a flat elect rophoretic kind shown in Fi'ure 12. N . mndsry FAectrophoresis {m.s x. x has the hhest and z ha the lowet mob.427 0ntained in a closed system composed of a thin-walled section for optical observ ations and of table electrode compartments. X÷ +Z Buffer ÷ Prote/v. yield electrophoretic patterns that sho w the direction and relative rate of migration of the major molecules in the sample.GIobulkn Globulin " I l I glectrophoretic mobility . they migrate from the macromolecule solution t o the pure buffer or into the zone of macromolecule free buffer and form a boundary or fron t. As they do so. The pH of the buffer is.428 Biophysical Chemistry In moving boundary electrophoresis a buffered solution of macromolecules is plat ed under a layer of pure buffer solution in a U-shaped observation cell (Figure 12. The movement of macromolecules consequent to generation of electric field between the electrodes will then be towards the anode. Normally in a complex sample containing many macromolecules.5 A). Figure 12. when measured by appropriate optical devices (us ually Schleiren optics or Rayleigh interference optics). The whole cell may then be immersed in a constant temperature bath insulated from vibrations. electric field between the electrodes. therefore. This situation of movements in mu tually opposite directions will not be good for a satisfactory resolution. which will bear a net neg ative charge and therefore move towards the anode while at the same time the macromolecules b earing a net positive charge will move towards cathode. The refract ive index changes along the electrophoretic ceil. chosen that all the macromolecules bear a net negative charge. As a result of this there is sharp change in the refractive index of the solution at this bound ary (the index of refraction of macromolecules is different from that of pure buffer). Albumin -SpikeA ' II . T he power is switched on generating an. there will be species.5 B shows such a pattern. It. it has been superceded by techniques collectively known as zone electrophoresis. ZONE ELECTROPHORESI$ The same year (1937) that Tisclius described his moving boundary electrophorcsis . inexpensive.e. For many years moving boundary method was very popular for quantitative analysis of complex mixtures of macromolecules. 2. those in blood pla sma. Zone electropho resis is the name given to the separation technique employing these stabilizing medi It is al so known as . and polyacrylamide. Konig published the first experiment on the use of idter paper as stabilizing medium i n electrophoresis.ljw'e 12. however.g. This paved the way for several other porous stab ilizing media. routine technique..5 (B) Representation of an electrophoretlc pattern of serum proteins ob tained by botJlanj elec-trophoresis. In recent years. took ten more years for filter paper electrophoresis to become popu lar as an efficient. especially proteins. most of which are gels such as agar. however. starch. separation the molecules are Immobilized by fixation in different zones. the migration of can be observed..e.x Y 429 tn stabItr. the components can be eluted from the medium and thus become available for further Zone electrophoresis can also be utItlzed as large-scale or preparative method w hereby amounts of a component can be purified for further characterization. In boundary separation. The mol ecules detected by staining them on the supporting medium. . .ed media. (tO detection by virtue of enzymic or (t) detection by radioactivity. x 'y A comparskn ofseparatlon obtained with (A)free electrophoresla and (B) zone elec trophoresis. As a result convection currents pose a big problem. ff the molecules are radiolabeled. Alternativ ely. The only can be isolated in substance are the slowest and the fastest migrating other intermediate components overlap and can only be observed as separated but be isolated. As t he progresses. mobility and lead to an inadequate separation. Other methods to detect the molecules are (/) visualization by ultraviolet light. i Comparison of Free and Zone Electrophoresis Figure 12. X and Y are two components resolved by the two technklues. The separation is carried out usually in a 'U' tube with the sa mple i layered under a buffer solution. in free electrophoresis. more heat isproduced and the convection currents increase. A great advantage of this analytical tool lies in the fact that are often quite sufficient for a complete electrophoretlc separation.6 depicts the difference between the separations that can be obtained with free zone electrophoresis. The system does not use any stabilizing mediu m but is the density difference. The zones have higher density the medium. However.The zone separation uses stabilizing media (paper. but the Use of stabfllzing system does not allow the zones to disper se and as is the case with free electrophoresis. gels such as starch. zone electrophoresis i s a much usehfl tool than the free electrophoresis. The stabillzlng media might influence and isoelectric points of sample long in a very unpredictable manner. each zone consist ing princlple). . etc. the sample components during their migration spl it up different zones as it contains differently grating components.).which can be easily isolated. polyacry lamide. i f is separation and not a Study of electrical properties. In zone electrophoresls. The two buffer reservoirs are usually each partitioned into two interconnected one cohtaining the electrodes and the other in contact with the supporting mediu m. Wicks are made up of filter paper or gauze. The power pack provides a stabilized for both voltage and current output. the paper can be made to dip at both the ends thereby obviating the need for wicks. buffer reservoirs. The paper is placed on an material. The supporting medium i s with the buffer prior to the start of electrophoretic process. Power packs. PAPER ELECTROPHORESIS Filter Paper Filter paper as a stabilizing medium is very popular for the study of normal abnormal plasma proteins. The whole electrophoresis unit is then covere . GENERAL TECHNIQUES OF ZONE ELECTROPHORESIS 1.430 Biophysical The apparatus for moving boundary electrophoresis itself and the associa ted optics are very costly. as a preparative technique is logical. Chromatography paper is suit able electrophoresis and needs no preparation other than to be cut to size. Paper of good quality should contain afleast 95% of ce llulose should have only a very slight adsorption capacity. a support a transparent insulating cover. Apparatus The equipment required for electrophoresis consists basically of two items. which have an output of 0-500 V and ( mA are available and can be programmed to give either constant voltage or curren t. The free electrophoresis on the other han d cannot modified for the purpose. Altematively. a po wer and an electrophoretic cell. as it is difficult to isolate individual components of a sample by method. usually a perspex sheet. The electrophoretic cell contains the electrodes. This is another restriction for the the apparatus for zone electrophoresis is simple and cheaper. As the zone separation resolves the sample components in a much better manner. The electrodes are usually made of platinum. compartments are necessary so that any change in pH occurring at the electrodes does affect the buffer in contact with the supporting medium. 5 cm in diameter) or as a narrow uniform strea k. There is no hard and fast of application of the sample to the electrophoretogram. Sample Application This is the single most critical procedure in the whole electrophoresis process. The horizontal and arrangements (Figure 12. Commonly used buffers are listed in Table 12. Although deices providing stable voltage or current are available. Two arrangements of the filter strips are colnmonly used.d with insulating material to minimize evaporation during the run and to insulation.7). This can be applied befo re the has been equilibrated with the buffer or after it. the equipment has to b e intermittently throughout the run because overheating is a distinct posslbflity (if not checked . The may be applied as a spot (about 0. Eletrophoretic Run The current is switched on after the sample has been applied to the paper and th e has been equilibrated with the buffer. devices are available commercially for this purpose. Both the arrangements are equally viable and the cho ice depends upon personal preferences.1. . The is switched off after the run and before the paper is removed. I perspex cover / " I . The process .. Overheating can be avoided by placing th e entire in the cold room..o-i Bufe" Insulattng plate (A) Horizontal paper el'ophoresis Buffer Electrodes (B) Vertical paper electrophoresis Figure 12.Re. - I -.431 EI trophoretic " filter paper..- .l.usually does not take longer than two hours. 7 Paper electroptwresis heating may lead to charring of paper). 1 Buffers for Electrophoresis of Different Substances Biophysical Chemistry Intended Buffer Separation Proteins Mucoproteins Amino Acids Phosphate Barbiturate Citrate Phosphate Phosphate Michaelis .05 Barbital* Barbital Rheophor Phosphate Borate Acetate 432 Table 12.7 1/15M 0.7.I 0. 2 8.6 8.Nucleic acids Borate Tris acetate Tris phosphate pH Ionic Composition/Liter Strength (} 8.6 9.4 8.2 7.0 4.075 .05 0.6 7.6 8.4 9.6 4.5 4.6 7.8 0.5 4. 2. 20.05 g Barbiturate.3. 90 ml 0. 3.2 0.5 9. 1.6 g Na Barbiturate. 8. 3.0.68 g Barbital 28. IN HCI 3.05 7. adjust pH with acetic acid (5x stock solution) 21. 3.8 g Tris base.15 10.8 g Na Barbiturate. 3.6 g Citric acid 20. 23.6 g NaH2PO4.62g Boric acid.4 g NaHaPO.4 g KHPO 2. 0.44 g NaHPO4.2 g Na acetate.g Na borate 24.9 g Na acetate.76 g Barbital 10. 60 ml 0.13 0.3 g Na Barbiturate.51 g NaCl.196 g NaH2PO.2 g Na2HPO4 Na borate.I 0. 2.2HO.46 g Na citrate.3 g Citric acid 20.45 g Na barbiturate. I 0.25 g Na acetate pH adjusted with N HCI to 4.622 g NaHPO4-2H20 9. 10.H20.2HO. 8.63 0. . 1.1N HCI 0.84 g Barbital 15.85 g Na2EDTA.1 g Tris base. the zones of resolved components spread. Many times during staining.85 g NaEDTA-2H20 (5x stock solution) Especially useful for hemoglobin. Physical proper ties like fluorescence. a . Proteins.1. Alternatively. ultraviolet absorption or radioactivity (if the sample has ben lab eled) are exploited for detection. Once removed. (/) F/uorescence. Dansyl chloride may also be used in pla ce of fluorescamine. Similarly fluorescamine staining is utilized for detecting amin o acids. (ii) Ultraviolet absorption. the electrophoretogram can be stained to convert t he resolved components into colored derivatives. Different dyes are used for detecting different components (Tabl e 12. and this property can be exploited to detect these components on an electrophoretogram. Staining with ethidium bromide and subsequent visualization of the electrophoretogram under ultraviolet light makes DNA and RNA fluoresce and thus facilitates their detection. the paper is dried in vacuum oven at 110°C (if the compounds are not thermolabfle. (ill) Staining. In this respect electrophoresis is similar to chromatography. amino acid derivatives.2). Detection and Quantitative Assay To identify unknown components in the resolved mixture the electrophoretogram ma y be compared with another electrophoretogram on which standard components have been electrophoresced under identical conditions. in which case the paper is allowed to hang and air dried). peptides and nucleic acids absorb inthe r an]ge of 260-280 nm. Individual compounds are usually detected and identified n sttu. As a safe m easure. peptides and proteins. aining procedure is mpleted.2 Visual and Fluorescent Dyes Used to Detect Components Separated by Iietrophoresis Polysaccharide General Bromophenol blue in acetic acid Nigrosine in trichloro acetic acid/ acetic acid Lissamine green in acetic acid Coomassie Brilliant Blue R-250 Dansyl Chloride Fluorescamlne Aqueous anilinonapthalene sulphonate (ANS) Methyl green-pyronine Lanthanum acetate + acridine orange in acetic acid Toluidine blue Pyronine Methylene blue Ethidium bromide Sudan Black in 60% ethanol ..Acids Proteins Llpoprotelns Comments 'trophoresis 433 xative may be applied before staining.. lble 12. Excess stain is eluted after st. Periodate oxidation + treatment with Schiffs reagent /Iclan blue Iodine Stains-All . is modified in such a way that it should become coloured upo n reaction . Proteins-red. RNA-orange red Visual.sensitlve Visual. quantitative Fluorescent. sensitive Fluorescent. quantitative Visual. quantitative Vlsua. visual. however. RNA-red sensitive DNA. senstive Visual. substrate. Phosphoproteins-blue. quantitative Fluorescent. sensitive RNA. A paper strip of the same size as the is impregnated with the substrate for the enzyme desired to be separated. sensitive RNA. Visual. special techniques may be used to detect it. very sensitive DNA-blue. very sensitive Visual. quantitative Visual. sensitive DNA-bluish purple. RNA-blue. If the component to be separated is an enzyme. very sensitive Visual. very sensitive Fluorescent. Acid mucopolysaccharides -blue to purple (iv)Detection of enzymes in situ. for accurate estimation the compound eluted from the electrophoretogram.of the zone and the resulting v alue would be a rough component. the color density of the . To impart more accuracy. After all the color has been extracted. A rough idea of the quantity of the components of a s ample had by visually comparing ("eyeballing") the color of different zones with their standards known quantity. However.the enzyme. The posit ion of the )earance of Color on the paper strip gives the position of the enzyme on the (Figure 12. After a particular time the two sheets are separated. But this is seldom accurate. (v) ant/tat/ve estimation. The paper strip is now juxtaposed with the electrophoretogram and pl aced in suitable buffer. For elutlon the zone is excised from the pap er and in a suitable solvent.8). the c61or density. of zone may be multiplied with the area. (D) Upon removo at a certainposition colorwould be observed on Ore subetratepape r. One if these bands is due to the desired enzyme. ing band on the electrophoretogram (in this case) is due to enzyme. If the sample was labeled before the run. T his gives fairly accurate idea of the quantity of the solute. solution is measured and compared with that of the standard of known quantity. Alternatively the zone can be excsed and p laced in scintillation vial and solid scintii/ation counting can be carried out. //ter papere. e. ardsol}. In some eases. (A) A given sample containing the enzyme has separated into four bands as tion. the paper is made transparent by immersion in organic refractive index (e. Sample may range from crude tissue fluid and extracts to the most highly purified protein preparations.. Example: Separation of Protein by Paper Electrophoreeie Sample preparation. Most body and tissue fluids are applied directly to the paper without diysis when small samples are used.S DetocOnn of enzyme in situ. The transparent paper is placed between two glass plates and absorption of the colored region ie measured by densitometer. the separated zone can be eluted and a scintillation cocktail and counted. . paraffin oil-bromonaphtha/ene.g.(C) Figure 12. Although a wide variety of papers can be used. in most clinical work serum is directly placed on t he paper.g. so/t thick papers h ave been found to give better resolution. The substrate is so modified that it will develop co lor reaction. (B) A paper sheet of the same size as the electrophoretogram is imprnated wi th a $bstrate the enzyme to be separated. (C) Substrateimpregnatedpaperjuxtaposadoverthelectrophoretogram. which gives an ideaabout the quantity of the resolved component. 6.1-0. One of the most commonly used dyes for staining proteins is bromophenol b lue. Best results. however. are obtained at a pH on the alkal/ne side of th e isoelectric points of the proteins in the mixture.. as a rule. Excess dye cn be washed offunder running water. 0. tInOW.2). and (//) veronal/acetate buffer at pH 8. The sample in small quantity (e. The paper is dipped in 0. I. The power is switched on. The origin is marked on the paper with the help of a pencil. . Once the run is complete. the papers are dried m an air oven at 100°C. Subsequen tly.g.1% bromophenol blue solution in ethanol saturated with mercuric chlo ride..05 M veronal bu ffer at pH 8. Alternatively coomass/e blue can be used (Table 1 2. Teeha/qu. Usually 14-16 hours are required for sepa rat/on at 100-200 volts.6 and ionic strength 0. It takes about 30 minutes for the paper to equilibrate with the electrolyte. The commonly used buffers include (i) 0. the paper is placed on an insulting solid support with its ends hanging in the electrolyte (wicks ca n be used to establish contact). If.4 ml of serum} is applied on the origi n either as a spot or a streak. a higher voltage of th e order 400 volts may be used and the time of separation gets reduced proportionally (the risk of overheating is p ractically nil in a cold room)./ffere. the separation is being carried out in cold. The paper is allowed to dip int o a saturated dye solution in methanol and 10% acetic acid. Alternatively naphthalene black 1213200 is used. . The paper is kept in the dye solution (100 ml dye sol ution contains 50ml methanol. Additional advantages of cellulose acetate are (/)it is chemically pure. Eluting the colored zone and performing colorimetry of the extracted c olor can ive the most accurate idea of the quantity. Serum analysis for disgnostic purposes is routinely carried out by paper electro phoresis. t/ e/mtn. milk proteins and snake and insect venoms etc.Electrophoresis 435 Azocarmine B is also used. which are thin and have a uniform micro pore ' . .Although paper electrophoresis is still the choice for routine fast diagnostic analyses. (//} cellulose strips are translucent and this makes them for directhotoelectric scanning for separated bands of components. CFLULOE ACETATE ELECTROPHORESIS Cellulose acetate as a medium for electrophoresls was introduced by Kohn in 1958 . Other proteins that have been satisfactorily analysed using paper electrophoresis include muscl e protein. Each colored zone is cut into 5mm wi de strips at right angles to the direction of separation and placed into a trough containing about 5ml NaCO3 solu tion (1part . egg white proteins.10 % aqueous NaCO3 solution). It was developed from bacteriological cellulose acetate membrane filters and is commercially as high purity cellulose acetate strips.this solven t is sufciet and the resultant color is measured in colorimeter. the resolution of a given protein might suffer because of substantial adsorption on paper.methanol and 1 pert. About 30 minutes of extraction in. (iii) be. 40ml aqueous solution of azocarmine B and 10ml of 5%acetic acid) for I 0 minutes and subsequently washed for 5 minutes each in methanol and I0 % acetic acid. it does not contain hemicelluloses or nitrogen. This paper is completely taken care of if cellulose acetate strips are used instead o f paper. 2.caus e of very low content of glucose these strips are suitable for Ciectrophoresis of pol ysaccharides. After 10 minutes the paper is washed s everal times with methanol containing 10% acetic acid. . Cellulose acetate is not suitable for preparative electrophoresls.thus holds very littie buffer.he pore and be retarded or will bypass it.Needless to say that resolution of a sharper and better in a gel than in any other type of medium. lipoproteins an d from blood. The gels. The strips are suitable for immuno-electrophoresis (see later) and Cellulose acetate is not very hydrophih'c end . Solvents rendering the strips transparent are preferred rather staining agents. background staining is negligible. I). Such solvents are glacial acetic acid. The buffers used for cellulose acetate electrophoresis are essentially the same as used for 12. The separation thus not only the chsrge on the molecule but also on its size. is conducive for a better resolution in a shorter time. Cellulose acetate is especiaJly for clinical investigations such as separation of glycoproteins. are porous and the Size of the pores relative to that of the molecule determines whether t. is responsible for greater heat production and the electrophOresls has to preformed. liquid paraffin or a special medium sold by cellulose strip manufacturers. Whitmore oil 120. 0 oa sxs In all the types of electrophoreses we have discussed so far (free electrophores ls. The of the strip helps in direct photoelectric quantitative determination of the without elution being involved. Thi lower buffer capacit y however. cotton seed oil..Schs reagent. . paper cellulose acetate electrophoresis ) cmrge on the molecule was the major determin ant for its mobility and ultimate separation from the rest of the molecules. removed by purification. the starch gel tu rns opaque making direct photoelectric determination impossible. One of their important applications is the analysis of isocnzymc patterns (zymograms). The pore size in a starch gel cannot be controlled and this is the bigge st drawback of these gels. it is difficult to prevent contamination of starch gels by microorganisms. Mor eover. wherein temperature control and the timing of hydrolysis are extremely important .Acrylamide 436 Biophysical Chemistr (i) Starch uel. The sulphate content is however. Starch gel as a stabilizing medium for zone electrophoresis was introduced by Smithies in 1955. The suspension is then neutralized with sodium acetate and washed with large amount of distilled water and dried with acetone. Agar solubilizcs in aqueous buffers above 40°C and sets to form a gel at about 38°C. the resolving pow er of starch gels is very high and can be matched only by polyacaj1arnide gels. which precipitates the agarose. Starch gels are unsatisfactory for separation of basic proteins. these gels have low diffusion resist ance and are . q/) Ar. Agar gel electrophoresis was first described by Gordon in 1949 and has b een since used increasingly for biochemical separations. The latter is collected. Another disadvantage of starch gels is that upon staining to detect the separated components. The latter is sulphated (and thus is charged) and therefore may give rise to severe electroosmosis which will be detrimental for electrophor etic separation. washed with distilled water and dried with acetone. agarose and agaropectin. Potato starch is hydrolyzed in acidified acetone at 37°C. Since the molecular sieving action is negligible.-However. This hydrolyzed starch when heated and cooled in a n appropriate buffer sets as a gel (the amylopectin chains intertwine). High porosity starch g els are obtained by using 2% (w/v) starch and low porosity gels are obtained by adding I0-15% sta rch to the buffer. The process is re peated three times and the agarose so obtained is virtually free of sulphate. A low quantity of agar in buffer gives a large pore size and almost no molecular sievi ng action. A solution of agar at 80°C is mixed with an equal volume of 40% polyethylene glycol. Agar consists of two galact ose-based polymers. The most comm only used components to synthesize the matrix are acrylamide monomer.consequendy of much use in immunoelcctrophoresis for detection of antigenic prot eins.Methylene-his (acrylamlde) Figure 12. Agar is being used to separate high molecular weight macromolcculcs like proteins and nu cleic acids.9. N'-methylencbisac rylamide (his). ammonium persulphate and tetramethylenediaminc (TEMED).9 Components of polyacrylamide gel Ammonium pcrsulphate when dissolved in water generates free radicals. . In the latter case its use has become legion. The components used in the formation of this gel are known to be neurotoxins and thus care has to be taken while preparing the gel. CHa N--CH2---CH---N< HsC CH3 Tetramethylenedlamlne {TEMED} O O II II CH = CH--C--NH--CH2--NH--C--CH = CH2 N. N'. CH2 :ICH--C--NH2 HaC . (/i) /rmd. N. Chemical structur es of these compounds are shown in Figure 12. xist in free radical . H ONH2 SO " (persulphate) CH2 -. For example riboflavin may be added to acrylamlde.+ CH2(CH2 = CH---C--NH)2 -Acrylamide N. TEMED acts as a catalyst of gel formation because of i ts ability to . The whole reaction is shown in Figure 12.form.CI-.Electrophoress 43 7 These free radicals can activate acrylamide monomers inducing them to react with other acrlamide molecules forming long chains in the process.-CH. which is dependent upon the .CH-CI.Methylene-his (acrylamide) --CH. H.CH. Riboflavin undergoes photodecomposition and forms free radicals which act much in the same way descri bed above for ammonium persulphate.CH-.O H J CH2.10. I0 ReactWns fnvolved fn polyacrylamfde get formatlon The pore size of the gel is determined by the amount of acrylamide used per unit volume of the reaction medium and the degree of cross linkage. bisacrylamide mixt ure and the whole mixture then irradiated with ultraviolet ]ight in presence of oxygen. I CON-H' CONH I --CH2--CH--CI-I2Fgure 12. N" . N'-methylenebisacrylamlde. ..-CH-.--HCH2--CH--CH2CH-'CH2 CONH] c. These chains become cros s-linked if the reaction is carried out in the presence of N. Ammonium persulphate and TEMED can be replaced by o ther compounds.-H-.-. Other desirable featur es of these gels are (f) a low adsorption capacity.amount of bisacrylamide used (see Box 12. and (f/0 suitabil ity for in sltu histochemical and qualitative analysis. (f0 lack of electroosmosis. This feature makes this gel particularly suitable for resol ving mixtures of proteins and nucleic acids in a very reproducible manner. I). The pore size can be controlled by controlli ng the amount of these two compounds and a relationship between the concentrations of the two and pore size has been arrived at. . 5%. which have been used include pectin. The acrylamide used m ay be as low as 0. logE = log E'. agarose provides physical sup port to the low percentage acrylamide gel which provides the molecular sieving action. The need to separate very high molecular weight nucleic acids (200 Kd or more) prompted the development of this complex gel.438 Biophysical Chemistry (/v) A-Aerlam/de.5% only.5% agarose in bofllng water and allowing it to cool to 40C. polyvinyl chloride and polyvinyl acetate. co mponents needed for polyacrylamide gel formation are added and the mixture is transferred to eit her a colunm or a slab gel rig.5 x 10 daltons DNA.that of the s olution. Agarose acrylamlde gels are prepared by dissolvi ng 0. thus the molecular sieving action and therefore the effect on ele ctrophoretic mobility of a molecule are functions of gel concentration. Rodbard and Chrambach have developed a set of mathematical relationships to describe the effects of gel concentration upon a macromolecule's mobility. In a mixed gel. Electrophoretic Mobility in Gels The discussion on electrophoretic mobility in previous pages did not take into a ccount the molecular sieving action of the gelsand its effect on the mobility of a macromol ecule. Acrylamide gels with pores large enough for these large molecules remained liquid because the quantit y of acrylamide was 1-2. which has been brought to the same temperature as . E' is the mobility in a sucrose solutio n. however. Sephadex. on the other hand. (v) Other gels. Kr is the retardation coefficient and G is the total gel concentration. The retardation co efficient has been described as follows Kr=C(R+r) . gypsum. The pore size. At this temperature. However. they are not used often and hence a discussion i s avoided. The gels were first used by Peacock and Dingman in 1968. Agarose alone. The gel sets on cooling yielding a highly porous and rigid matrix.K where E is the electrophoretlc mobility. has no molecular sieving acti on necessary for a sharper resolution. These gels have been successfully tried for isolation of 3. .-or by hydrophobic associations. however. disulphide bridges. These proteins migr ate as a single band during gel electrophoresis.where C is constant. The whole structure is stabfllz ed by hydrogen bending. 6. These proteins are referred to as oligomeric proteins. . a class of substances known to destabilize the quaternary struc ture are employed. a. and 6' but migrates as a single specie if subjected to zone elec trophdresis. the subunits of these proteins are to be separated from each other. R is the mean radius of the macromolecule and r is the radi us of the gel fibers. Several proteins of biological importance contain more than one polypeptide chai n. If. This enzyme has three n onidentical subunits. An example th at can be cited to illustrate the point is of core RNA polymerase. These substances are collectively known as solubil.ers. haptoglobins. Urea at concentration from 3-12 M is known to disrupt hydrogen bonds.. if treated with SDS (see below). The differential migration of these macromole cules is then a index of their molecular weight and is empirically used for determination of rel ative molecular mass (see applications of gel electrophoresis).coll ribosomal proteins and a-crystallin. and electrophore sced on a gel in which SDS has been incorporated. disassociate readily in concentrated urea. . (i) Urea. a-and -caseins. the three polypeptide chains migrate as three d ifferent bands. the structure of which is solely m aintained by hydrogen bonding. Since all the polypeptide chains now have an equal negative charge due to SDS. The principles remain parallel to those dexcribed for SDS. SDS has been shown to bind to the hydrophobic regions of proteins and to separate most of them into their component subunits unless the subunits a re covalenfly bound. Thus those macromolecular complexes or aggregates. Cetyltrimethylammonium bromide (CTAB) is used as a cationic detergent in place o f SDS ardonic. Often used solubfllzers are discussed below.permitted a number of separations not achieved by any other m ethod. has thus. (CH3(CH2)oCH2OSO Na÷).Electrophoresis 439 However. In addition. a solubfllzing agent. Thus the presence of urea in the gel in which electrophoresis is conducted is essential. SDS binding also imparts a large negative charge to the denatured polypeptides. they will migr ate in gel solely on the basis of their size. y-globulin light and heavy chains. Use of gels containing substances capable of solubilizing certain macromolecules . Certain examples where use of urea ha s provided additional information about the macromolecular structure are myeloma y-globulin . double stranded DNA can be rendered single stranded by use of urea. mainly proteins. (il) Sodium dodecyl sulphate. re moval of urea may be expected to lead to reaggregatioti. In such systems. E. Sodium dodecyl sulphate (SDS) is an anionic detergent and disrupts macromolecules whose structure has been st abilized by hydrophobic associations. This charge shadows any other charge previously present on the polypeptide. The whole may be covered by a perspex shield. These bonds are broken by heating the protein so lution in of mercaptoethanol. The dye migrates faster than all macromolecules. A 'trac/ctng dye' ( usually ) is often mixed with the sample. acetic acid and water (2:1:1) ha s been used.(iii) -mercaptoethanol. prepared in a high density component such as glycerol or flcoll to p revent its with the upper reservoir buffer is loaded on top of the gel. Save for the gel. Platinum electrodes are positi oned reservoir and are connected to terminals extending from the top of the unit. Thu s if the . This solvent . The sample. and (ii) a D.of the equipment is shown in Figure 12. Note: When protelns are insoluble in a concentrated aqueous solutlon of urea. A suitable alkylating agent like iodoacetate is also incorpo rated reformation of disulphide bond. The buffer reser voir buffer reservoir connected by the gel. Phenol. system has been used to separate proteins of ribosome sub-units. The extent of migration of the dye gives an of electrophoretic process. Procedure The equipment consists of two components (/) a buffer reservoir.11. electrophoresis may be possible in solvent systems of a different type. there is electrical connection between the two reservoirs. Many proteins have their plural polypeptide chains linke d together by disulphide bridges.C power A schematic diagram . 11). They are essentially the electrolysis of. When fle power is switched on. CATHODE REACTIONS 2e. to which these negatively charged macromolecules woul d migrate under an electric field. a diligent observer will see that the amount of b ubbles generated in the reservoir containing the anode is much less than that in the re servoir containing the cathode.A. The anode. Also shown are the Modes of Gel Electrophoresis . However.1 I). The bufferis allowed t o enter drop by drop to avoid short circuit. The reason for this lies in the reactions that permit the flow of c urrent from cathode to anode. drop by drop (Figure 12.11 Schematic dlagram of an electrophoretlc system with buffer recyding s ystem.. 2OH.440 Biophysical Chemistry electrophoresis is stopped before or just as the dye comes out of the bottom of the gel. is therefore placed in the lower buffer reservo ir. for each mole of hydro gen produced at the cathode only one-half mole of oxygen is produced at the anode leading to less number of bubbles being seen.water producing hydr ogen at the cathode and oxygen at fle anode (Figure 12. again. If the electrophoresis is going to take a long time it is necessary to replenish the diminishing level of buffer in the upper reservoir. one can be reasonably sure that all macromolecules are still within the gel.+ I-IO Upper I i Psupply 0 2H*+O+2e-H lure 12.+ 21-IO . This is done by recirculating the buffer from the lower reservoir to the upper reservoir with a peristaltic pump. which gives a net negative charge to most macromolecules.. The pH is usually fixed at 9. An over flow tube is positioned in the upper reserv oir which prevents an undue increase in volume and the extra buffer flows back to the lower reservo ir.+ H HA + OH. 2. Other modes are given in Box 1 2.Gel electrophoresis is usually carried out in any one of the two modes (0 column electrophoresis. or (//) s/ab gel electrophoress. . The gel is set or polymerized in a column.12(A). This column i s then between the upper and the lower buffer reservoirs.441 Electrophorests Column electrophoresls. An apparatus is commercially available has between 8-12 columns fitted into buffer reservoirs (Figure 12. . Slab gel electrophoresis.A column i s shown n the foreground.442 Figure 12.12(A) Photograph of a Colurnn gel electrophoresis apparatus. The gel is set or polymerized into a thin slab between two plates. The thickness of the slab of the gel can be adjusted by placing spa cers of various Upper Buffer Reservoir Gel between Two Plates Lower Buffer Resewoir . gel becomes opaque after staining. After the gel has set. This makes it difficult for direct densitomet ry to estimate the quantity of a component. Sample wells are made at one end of the gel by placing a comb-shaped jig into the gel before it sets or polymerizes. polyacrylamide absorbs in the UV range. allowlng the gels to be extracted under gentle pressure.Electrophoresis 443 thickness between the two glass plates. The technique is becoming extremely popular.3) Gels from a column are removed by forcing water from a hypodermic syringe around the of the column. . If one desires accurate quantitative analysis or requires the separated componen ts for the compound has to be removed from the supporting matrix. Compound from starch gel can be removed by slicing the appropriate portion of the gel. This is a great advantage of this technique over the column mode.12(B)).simultaneously and co mpared under conditions which remain essentially identical. The same stains as described in Table 12. thus some background absorbance. number of samples can be loaded. Sinee a n umber of wells ican be east side-by-side. The problem may be obviated by treating stained gel with glycerol and acetic acid which renders it transparent.2 are used for staining components in g els. the comb is removed leavingthe sample wells etched into the gel (Figure 12. Slab gel s are by introducing a thin metal plate between the two gel plates and coaxing the pla tes Before staining the gels may be immersed in a fixative (7% acetic acid) to guard against of separated components. However. especially in the field of molecular Recovery and Estimation The principles and major methods for detection and estimation of separated compo nents the same as described for paper electrophoresis previously.. Only those points sa lient to below (also see Box 12. Direct u ltraviolet may also be performed. macerat ing it and it with amylase which solubfllzes the starch and leaves the compound in solution . Radioactive compounds can be eluted into vials containing a suitable scintillati on cocktail Ghapter 13) for counting. Alternatively it can be put in a small Eppendorf tube small holes. The gels are however. If the sample has been radioactively labeled prior to electrophoresis. . autoradio graphy may performed to detect the position of the separated components in the gel. The rubbery constit ution of the forbids any attempt to macerate it. The sla b or tubular in a thin polythene sheet and is placed in a folder and exposed to a X-ray film. The small tube is then put in a larger Eppendorf tube and microfuge d for a at high speed. pretreated with H202 at 60°C to el iminate quenching by gel polymers. may then be solubillzed in appropriate solvents.acrylamide gel is frozen and then cut at the requisite size. The gel strip is put into a syringe and extr uded a thin gauge needle with force. The gel is extruded through the holes into the larger Eppendorf t ube. 444 Biophysical Chemistry . electrophoresis is carried out in the s o-called denaturing media. to get a reliable data about the molecular weigh t of it is necessary to eliminate the conformatlonal differences by destruction of th e base pairing. but mol ecular weight (see later).Gel Electrophoresis of Nucleic Acids: FAeetrophoresis in Denaturing Media Although. Thus all RNAs do not adhere to the e molecular weight calibration and gel dectrophoresis in conventional media migh t provide erroneous data of many RNA species. Other reagents . Although charge to mass ratio for a given RNA strand is always cons tant. since it will interactions between adjoining bases). The formamide method has been successfully used for mol ecular weight of many RNA species. The supporting electrolyte may be sodium chlor ide without any or barbital may be used. first such experiment tried formaldehyde as the denaturing agent. all the basic features described above for gel electrophoresis apply e qually well for separation of acids. which have been used : 8M urea and formamide. Another situation which calls for electrophorcsis in denaturing media is when th e objective of the . that even a chain devoid of base pairing will not be entirely structure less. Therefore. To eliminate conformational differences. The latter is more frequently used since it has been fo und to allow unequivocal of molecular weights with considerable accuracy. conformatlonal ' arise between RNAs of'similar molecular mass. (It has to be however. In this technique formamide ser ves as the polymerization of the acrylamide. These differences are mainly due to the random and base pairing of RNA molecule giving regions of double helix alternating with the region of single conformatlonal differences between similar molecular weight RNA strands give ris e to frictional in a gel and are reflected in electrophoretlc mobility anomalies. problem arises when the a/m of electrophoresis is not separation. of the DNA. Single stands are required for sequencing operations and this is ach ieved urea in the gel (SIN). Formamide is not popular for this application. Of Gel llectrophoresis Apart from separation and isolation of a large number of protein and other macro molecules this technique has been of immense use, and is still proving its indispensabilit y, gel is utilized for a larger number of analytical applications. The technique has of such use in molecular biology that one can not think of any molecular biology ,.at some stage gel electrophoresis will not be carried out. Some of the analyti cal of this technique are discussed below. in Molecular Biology (O Determination of DNA sequences: The two methods of DNA sequencing (Maxam and technique, and dideoxy nucleotide technique of Sanger} currently in use are both high resolution polyacrylamide gel electrophoresis. Both these techniques have basis. They both depend on generating specific sets of radiolabelIed fragments, each at a particular base. The use of high resolution polyacrylamide gels then allows differing by only a single nucleotide to be resolved as a distinct band and the sequence Southern and Northern blotting'. The complementarity of the two strands of DNA to find whether the DNA or the mutation in DNA in which we is present in the sample or not. If the sequence of a portion of the desired DNA is one can synthetically prepare a complimentary oligonueleotide, radiolabel it, an d make react with the sample which has been separated by agarose gel electrophoresis an d 446 Biophysical transferred onto a nitrocellulose paper. The paper can then be autoradiographed. Retention radioactivity onto the paper shows us whether or not the sequence we are interes ted in present. As can be seen, gel eleCtrophoresis is central to this theme of hybridi zation. It central to northern blotting which is concerned with RNA rather than DNA. (ilO l¢trtetton mapping of DNA: During the study of genomic or cloned DNA it essential to have some sort of map to differentiate one area from another. One of mapping is known as restriction mapp/ng, and the use of electrophoresis is, a gain, it. In its simplest form, the technique consists of (A) digesting the DNA with d ifferent endonucleases. (B) separating the resulting nucleotide fragments by agarose or polyacrytamide gel electrophoresis, (C) visualizing these fragments, while they are in the gel, ethidium bromide staining, and (D) estimating their size in relation to the stan dards run on' gel simultaneously. (iv) Such important techniques as DNA footpr/n/ng (to find out the regions.of DN A interact with proteins) and restriction fragment length polymorphism (RFLP, used to mutations in various genes in carcinogenesis and other diseases) axe dependent o n electrophoresis in as much as that the final analysis is done on the gels after has been carried out. (v) Among the most-successful uses of analytical gels has been the detection of molecules, which are processed (degraded) to give rise to the product mature spe cies. precursors differ very little in molecular weight from their products and the gel is the decisive factor in the experiment. Precursors of t-RNA, r-RNA and m-RNA have been observed by their different mobil]ties on high-resolution gels. (vi)The sensitivity of gel electrophoresls to variations in conformation of nucl eic acids also section on electrophoresis in denaturin4 media) has not only been applied t o nucleic acid molecules, but also been used to study the kinetics of interconvers ions conformation of many t-RNAs. . Applieatimm in Protein Study "k 5 glutamic dehydrogenase , fumarase x 4 [ "glyceraldehyde phosphate .Mobility Figure 12.13 Illustration of llneartty of the plot of electrophoretic mobfly of vat.us SDproteba comp/es to the of the moeu we@hts. (t) Determination of stoichiometry: The sub1 stoichiometry of an oli protein might be determined by electrophoresis of the SDScomplex after covalent been introduced into the protein. reagent dlmethyll suberlmidate NH = (OCH3)--(CH2)e---(CH30)C = NH is to produce cross ', residues. Most of the cross-] between lysyl residues will intrachatn, but some can be to be inter-chain. Ift is now electrophoresced (not orfly even cellulose acetate has been tried with good different electrophoretic bands will observed depending on the stoichiometry of the protein. that the protein is polypeptide chains. One would 447 corresponding to the monomer, dimer, trimer, tetramer, pentamer, and the hexamer . the absence of the cross-llnking agent one would observe only the monomer band u pon with SDS. This simple procedure is extensively used to determine the subunit of oligomeric proteins. i (li)Determination of molecular weight of proteins by gel electrophoresis: geviously (see section on solubilizers] SDS can be used to dissociate oligomeric proteins into individual polypeptide chains. The degree of SDS-protein binding is very high (a bout 1.4 i protein), and thus the charge on the .SDS-protein compl.ex is almost entirely due to the sulfate long. It can therefore be said that the surface charge of this complex p er unit regardless of the charge of the individual polypeptide chains. Moreover, it has complexes tend to assume the shape of a long rod whose width is the length of such complexes, therefore, becomes a function of the molecular wei ght polypeptide portion. These factors are at the base of.molecular weight determina tion by electrophoresis. It has been amply demonstrated (largely due to the initial work by and by Weber and Osborn) that in gels which exert satisfactory molecular sieving , exists between the electrophoretic mobility of an SDS-protein complex the logarithm of the molecular weight of the protein. The method has gained extr eme ' because of its simplicity, reproducible results, and easy interpretation; Figu re 12.13 the linearlty of electrophoretic mobility of SDS-protein complexes to the log of molecular weights. Molecular weight of quite a few proteins has been determined by this See Box 12.4 for some problems and answers regarding SDS-PAGE. SPECIALIZED EROPHORETIC TECHNIQUF. I. DISCONTINUOUS (DISC) GEL EOPHORESIS In zone electrophoretlc techniques described so far, even ff all the sample over the gel, the sample can never be loaded in a sharp band and sample impedes in a sharp resolution of the components. Disc gel electrophoresis because of the discontinuous buffer employed and discoid appearance of the zones) is a modification of conventional zone electrophoresis, which allows the sample to the gel as a sharp band, thereby helping further resolution. be analyzed is subjected to an electric field in a retarding gel support that is separated sections differing in porosity and buffered at different pHs. The macromolecular m/xture from the more porous into the less porous gel, a process accompanied by a change in pH. result, each macromolecular species becomes concentrated into a very thin, sharp . producing much higher resolution than can be achieved in a continuous buffer. The gel that is preferred for disc gel electrophoresis is polyacrylamide. The tw o porosity gels used are known as the stacking gel (high porosity) and separating (low porosity). The two gel system is illustrated in Flgure 12.14. 449 Chloride long Stage Current on sample in stacking gel 'Stage Late stage of electrophoresls; protein separated in many dlscold bands. Trailing long in running gel Glycine Sample Figure 12.14 Schematic diagram of disc gel electrophorests showing migration of lons at vaous stages. Gel The lower, separating or running gel is pre'ared using about 5-10% acrylamide wh ich is than that used in the stacking gel (the amount of acrylamide used depends upon molecular weight of the macromoleculelbeing separated; Table 12.3 provides about the differing compositions of the separating gel). Consequently the pores are and of a smaller diameter imparting molecular sieving property to this gel. It i s in gel that the macromolecules subsequently separate. The buffer used in this gel i s usually amine such as Tris, which is adjusted to the proper pH (Le., 8.3) using hydrochl oric acid. ', gel constitutes about two-thirds of the length of the column or the gel plate s. Prote/ns Table 12.3 Formulations fo Separating Depending Upon the Molecular Weights of th e Macromolecules Macromolecule Molecular weight of the Macromolecule to be separated .-yhmdde in sepm'stin I0,000 -- 40,000 40.000 n I00,000 I00,000 -- 300,000 300,000 n 500,000 > 500,000 15 20 I0 -15 5 -I0 5 7 2 m 4 Nucleic acids Oligomer I0,000 I0,000 -- 50,000 50,000 -- 200.000 200,000 2,000.000 15 20 I0 5 2.2 -- 3 Stcklg Gel After the separating gel has polymerized, a second layer of gel is polymerized o n top of it. This gel, known as the upper or stacking gel is prepared using about 2.3% acryla mide and is consequently highly porous and devoid of any molecular sieving action. The buffe r used here is also an amine, mostly Tris. The pH is adjusted with hydrochloric acid and is abo ut 2 pH units lower than that of the running gel (e.g., 6.7). The buffer used in the sample is identical to that used in the stacking gel. The buffer used in the lower reservoir is identical to that used in the lower or separating gel. The buffer used in the upper reservoir is also an amine. It however, differs significantly from the rest of the buffers in one important way. Its pH, which is kept slightly above that of the running g el, is adjusted not with hydrochloric acid, but with a weak acid whose pKa is at the desired pH. Glycine is commonly used for this adjustment. Electrophoretic Process Glycine in the upper buffer reservoir exists in two forms; as a zwitterion which does not have a net charge, and as a glycinate anion with a charge of minus one: ÷ NHsCH2COO- NH2H2COO-I-I Zwltter/on Negative charge When the power is switched on, chloride, protein, and glycinate anions begin to migrate toward the anode. Upon entering the stacking gel, the glycinate long encounter a condition of low pH (pH of the stacking gel buffer is about 2 pH units lower than that of the buffer in the upper reservoir) which shifts the equilibrium towards formation of zwitterions. As zwitterions donot possess a net charge, they are immobile. This immobility of glycine zwitte rions to migrate into the stacking gel coupled with high mobility of the chloride long creates a very high localized voltage gradient between the /ead/ng chloride and the tra///ng glycinate long. S ince proteins have their mobility intermediate between the trailing and the leading long, they carry the current in this region and migrate rapidly in this strong local electric field. The prot eins, however, cannot overtake the chloride long as the strong local field exists only between the chloride and the glycinate long. As a result the proteins migrate quickly until they reach th e region rich in chloride long and then drastically slow down. This two speed movement of the pro teins results Electrophoresis 451 in piling up of the protein sample in a tight, sharp disc between the glycinate and the chloride long. It is in this sharp band form that the macromolecules enter the running ge l. The smaller pores of the running gel retard the movement of the sharp band of the macromolec ules for a time long enough for the glycinate anions to catch up. Since the running gel pH is higher than that of the stacking gel the glycine long become fully charged again and the loc alized high voltage gradient disappears. From this point on, the separation of proteins take s place as in zone electrophoresis. But since the macromolecules enter the running gel as a sh arp band the further resolution in disc electrophoresis is sharper than conventional zone ele ctrophoresis. Proteases are enzymes which break peptide bonds. They are also known as peptldas es. Ther are mainly two kinds of peptidases m the exopeptidases and the endopeptidas es. exopeptidases, as the name indicates, break peptide bonds that are at the periph ery of the polypeptide -- if they break it from the amino terminal end they are called amin opeptidases, and if they break it from the carboxyl terminal end they are called carboxypepti dases. The endopeptidases break peptide bonds that lie in the interior of the polypeptl de. Some endopeptidases are very specific in cleaving peptide bonds. For example, th e Staphylococcus aureus V8 endopeptidase will break only that peptide bond whose c arbonyl function has been contributed by glutamic acid ( in other words the peptide bond must have a glutamic acid residue lying towards the amino terminal end); bovine pancreas ela stase will break only those peptide bonds whose carbonyl function is contributed by alanine , glycine, serine, or valine. Given the above, ttts conceivable that if one subjects different proteins to pro tease digestion, each of the proteins will be broken down into different number of fra gments of different sizes. If the protease digest is then made to migrate on polyacrylamide gel and subsequently the gel is stained for proteins, the pattern of bands will be different for differen t proteins. This is the prinle ofprotease mapp/ng. The banding pattern will be different not only for di fferent proteins, but also for the same protein with different proteases. Thus several different p rotease maps can be created for the same protein using proteases with different specificities. If you have an unknown protein on your hand, you can subject it to protease digestion of differ ent kinds and then compare the protease maps with protease maps of other known proteins. If th ere is a imatch, you have identified the unknown protein. Protease mapping is a convenient method of identifying proteins and polypeptides ' requiring no special equipment or technical facility beyond that necessary for S DS-polyacrylamide slab gel electrophoresis. The technique is performed mainly in two different way s. Suppose you have run a protein mixture on SDS-PAGE and have become interested in one of the bands that shows up after staining. A slice of this gel containing th e band is cut with the help of a dean blade. It is then equilibrated with stacking gel buffer, and set into a sample well of a second SDS gel. The gel slice is overlayered with protease in sample b uffer and electrophoresis is performed in a slab gel apparatus in the discontinuous system . Once the protease and the polypeptide are compressed into a band in the stacking gel, the power is stopped for a time. This is to give time for protease action. The protease actio n on the polypeptide substrates produces fragments that resolve into a pattern of bands once the powe r is switched on again. This pattern can be compared with a similarly treated reference sample in an adjacent slot on the slab gel. As said earlier, the banding, pattern varies with site spe cificity of the protease. The alternative method is to digest the polypeptide with protease in the sample buffer electrophoresis (not on the gel while doing electrophoresis as in the previous a lternative). is time consuming and burdensome. But this also gives rise to sharper bands and allows 454 Cathode (acidic) ZwRterlons Direction of migration of anions (A) Sample introduced at pH above isoelectrlc potnt sample components |anions) (C) Immobilized sample components at thetr Isoelectrlc 0') Mlgarttng sample components (cations) (A') Sample Introduced at pH below isoelectrlc point Direction of Increasing' migration pH of cations Flcjure 12.16 Schematic representation of the principle of Isoelectric focusslng Protein molecules may have a net positive charge in an acid solution because mos t amino groups carry a positive net charge and most carboxylic groups are protonated and electrically uncharged. With a gradual increase in pH, the number of carboxyl groups carrying a negative charge increases, while the number of positively charged groups decreases. At a certain pH value, the isoionic point, the net charge of the protein molecule is zero. The i soionic point of a molecule is thus determined by the number and types of protolytic groups and the ir dissociation constants. Although there is considerable variation in the isoionic point of pro teins (see Box 12.5), they are generally in the pH range of 3-11. In conventional elec trophoresis the pH between anode and cathode is constant and the positively charged long migrate towards the cRthode and negative long migrate to the anode. In lsoelectric focussing (also k nown as e/ectr focussbng}, on the other hand, a stable pH gradient is arranged; the pH increase s gradually from anode to cathode. A protein introduced into this jstem at a point where the pH is lower than its isolordc point will possess a net positive charge and will migrate in t he direction of cathode. Due to the presence of the pH gradient," the protein will migrate to an environment of successively higher pH values, which, in turn. will influence the ionization and net charge of the molecule. Finally, the protein will encounter a pH where its net charge is z ero and will stop migrating. This is the isoelectric point of the protein. The consequence of this is that every protein will migrate to and focus at its respective isoelectric point in a stabl e pH gradient, irrespective of its origin in the apparatus at the time the current was applied. Thus. the point of Electrophoresis 455 application and the volume of the protein solution are not critical. Diffusion, which is an obstacle with every other method of electrophoresis, is not a problem with electrofocussi ng, because focussing effect works against diffusion. Thus, once a final, stable focussing i s reached, the resolution will be retained even if the experiment is continued for a long time. The principle of isoelectric focusing is illustrated in Figure 12.16. R can be Elecphoress 45 Establishing the pH Gradient: Carrier Ampholytes The pH gradients may be obtained by electrofocusing special buffer substances kn own as carrr ampho/ytes. The carrier ampholytes must have the properties listed below. (0 Since carrier ampholytes must dictate the pH course, they should have a certain buffering capacity at their isoelectric point. (/ They should have a conductance at their isoelectric point. (///) They should have low molecular weight so that: macromolecules can be sep arated from them easily after electrofocusing. (/v) They should be soluble in water. This hydrophflic character will also pr event their binding to hydrophobic regions of proteins. (v) Ideally they should have a low light absorption at 280 nm. This would pe rmit the detection of proteins after electrofocusing by measurements at 280 nm. Carrier ampholytes are isomers and homologs of aliphatic polyamino polycarboxyli c acids. general formula for a carrier ampholyte is: R--N--(CH2).--N--(CH2)nOOH (CH2),---COOH,H, R his usually less than 5. Carrier ampholytes are available commercially in mixtur es covering a pH band (e.g., pH 3- I0) or various narrow bands (e.g., pH 5-6). Cona-nercial am pholytes include Ampho//ne (LKB), Pharma/yte (Pharmacia), and Bio-lyte (Bio Rad). The pH range of the carrier ampholytes should be chosen such that pI (isoelectric point) values of t he proteins r lie well within the corresponding pH range. When making a first run with a pro tein it is often advisable to work with the pH range 3- I0. Generally an amount of ca rrier which gives a final average concentration of l%(weight/volume) in the columns is For electrofocussing in gels, an average concentration of 2% (w/v) is reconmende d since electroendosmosis. When pH range is outside 6-8, ampholyte of up to 10% have been used in order to obtain a more even distribution of ' between electrodes. Convection - As for all other electrophoretic techniques described thus far, electrofocussi ng also needs stabilization of separating protein zones against convective flow in the solutio n. ways are in use: (/) density gradient, (//) gel, and (///) zone convection elect rofocussing. The is not as popular as the other two, but is discussed as a special case at the en d. (0 Density rad/ents'.Density gradients suitable for electrofocussing can be made with uncharged solutes, which are dissolvable in water to a concentration that will i ncrease sufflcienfly. The compounds should not react with proteins and should have a low metals. They should be of high purity. Sucrose is the most ideal compound for of density gradients as it has a protective action on proteins. It has been used with (w/v) as the densest solution. Maximum solute concentration and thus the maximum are placed at the bottom of the column. There is a linear decrease of the concen tration the solute.as a function of the column height giving rise to approximately linea r density However, nonlinear density gradients have also been used. Sucrose cannot, howeve r, at all pH ranges since it iS destabilized at pH range above pH I0. Glycerol is g enerally (c) Bophvstcal ChenUstry used at such pH rges. Oer compounds, which c be used foaflon ofdensl adlen, e mt ol, sorbitol, eylene ycol, r, d coll. (ti) , In electrofocussing, the gel sees only as an antlconvectant and not as a molecul sieve. Obously the gel concentraon should be low (7.5% for polyaclde) to prode lger dleter pores. For lge proteus, exceedg 00 , lower concenaon of aclde ght used in combaon th agose (0.5%). Aclde Is e prefeed gel for elecofocussg. os e sch gels e not prefeed as these gels pH gradient considerably dng prolonged ee ment. (No n ef. e appatus, lusated gure 12.17, Is de upof o rectl boxes, e upr one bei ng e cover. e upper surface of lower x is co--gated fldges, a het of about 10 , sepated by depressions, d is fa cing e upper box which has coesnd fldges. e fldges d depressions of e o hves fit teer leag a space of a few eters beeen. us from one end to e oer ere a now wave he chnel een the o ps wch c be descflbed as sees of tercoected broad U-tubes. e cer mphole soluon is led in this space odes situated at e two ends. Go e lid d the bottom p e hoow chelst into em ou e coolt quid ses to m a const temperate. en the cuent is on, a density adlent is foxed in each depression by e solute. en prots come iobile at the islc pH, e densi increases Iocy d e proteus settle do e depression of the bottom p. en e eeent is over, e cover is d e liqui d coects in e depressions. Each depression cont a fraction sepat by a dge. e cfl ons now cot out y ssib of conaflon by ne fracons. Cer phoa dg w cover ling chela Lid with .cpoling channels Ridge lalgure 12.17 Schematic dkgrarn of zone converctWn electrofocussu3 apparatus: (A ) Bottom partfllled with carrier ampholytes; (B) The lld with correspondl ridges; (C) The apparatus assembled for electrofocusstng. P! High pH Separation can be carried out in a vertical coluinn (Figure 12.18) or on a horiz ontal gel but in both cases purpose-made equipment is required. The column system is in mo dem superceded by the plate system. For preparative purposes, the column mode is sti ll glass colunms are commercially available. These are filled with mixture of carrier ampholytes suspended in density gradient solution (sucrose, g lycol, etc.). anode (upper) end of the colunm is connected to a reservoir containing an acidic solution phosphoric acid) and the cathode end is connected to a reservoir filled with an alkaline (e.g., sodium hydroxide). The valves of the reservoir are opened to allow the ac idic and alkaline solutions to diffuse through the column. This results in the formation of a pH the anode and the cathode. The valves are closed and the power is switched L. The carrier ampholytes now migrate until they become immobile upon reaching t he regions their corresponding isoelectric pH. These compounds remain fixed in these region s, and buffering capacity the pH gradient is stabilized. Now the sample is applied at end of the column. The charged components of the sample migrate in the electric their net charge becomes zero, Le., till they reach their isoelectric pH. The co mponent of the sample remain focused at the regions of their isoelectric pH. The whole p rocess take 1-3 days. Once the experiment is over, the power is switched off and the sa mple are allowed to run off through a valve at the base of the column into a fraction The fractions can be analysed further. C C 12.18 Schematic design of an isoelectrofocusing apparatus. (A) Anode platinum rug, (B) Cathode platinum ring, (C) Water jacket to maintain the temperature. (D) Power pack, and (E) Valv e to empty the colum The density gradient is shown by light and heavy shadowing. PI and P2 are two pr otein bands electrofocussed according to their isoelectrlc pH values. 460 Biophysical Chemistry Instead of density gradient, polyacrylamtde gel impregnated with carrier be used in the vertical columns. The time required for less than that required in the density gradient, {2-3 hours). The carrier amphol ytes are with the unpolymerized gel solution and the mixture is allowed to polymerize. Th e rest process remains the same as for the density gradient. Gels are, however, used mo re plate mode rather" than the column mode. This is so since up to 24 samples can b e analysed using the plate system. Separation of Protein" From Carrier Ampholytes The average MW of ampholytes is about 800 whereas that for a protein would be I0,000. This difference in the molecular weights immediately suggests two method s separating them from one another Dialysis against a buffer would effectively rem ove 99% of the ampholytes; but it is a slow process. Gel filtration would give effec tive se a very short time. Sephadex G-50 is the general choice for the process. Other methods for the purpose are ammonium sulphate precipitation of the proteins, ion chromatography and partition chromatography by countercurrent. Applications Electrofocussing has been widely used for separation and identification of serum proteins. It is being used by the food and agricultural industries, forensic and human laboratories, and for research in enzymology, immunology and membrane biochemist ry etc. 5. TWO DIMENSIONAL GEL ELECTROPHORESIS This powerful technique combines the resolving power of isoelectric focussing gel electrophoresis. The resolving power of the technique is so acute that it ca n resolw containing 5000 proteins into individual species. The mixture is first subjected to focussing on a I mm diameter gel in a capillary tube. At the the gel is extruded from the column and placed on top of a slab gel. The sample is now subjected to SOS acrylamide gel electrophoresis, which separates the proteins according to their molecular weights. Isoelectric point and molecular weight of a protein are in no way conne cted to each other and thus the technique exploits two properties of a protein for separation and thus has a great resolution power. 6. IMMUNOELECTROPHORESIS The technique exploits the specificity of reaction between an antigen and antibo dy and molecular sieving of the gel in which this reaction is taking place for analysis of components of a given sample. The technique was developed by Grabar and Williams and is actual ly a modification of Ouchterlony double diffusion technique. To understand the techni que better a brief discussion of Ouchterlony double diffusion technique is given below. The Ouchterlony method depends on the formation of a micellar structure during g elation of agar or sucrose. Molecular aggregates with molecular weights not exceeding 20 0,000 daltons can diffuse freely through micellar channels. The aggregates with higher molecul ar weights are, however, retarded. Thus, the individual antigen and antibody molecules filled in adjacent wells cut into the gel can diffuse freely till they come into contact. On contact, /f the ant/body/s specific forthe antigen, they form complexes with larger molecular weight; these complexes are immobile. If the concentrations of correspoding antigen and antibody are suffici ent to form a visible aggregate, a precipitin band appears (Figure 12.19). 461 Well for antiserum Wells for (c) ® ® Preclpttn band 12.19 Schematic Diagram of a precipltin band formed due to double diffusion, A n = Antigen, Ab -- Anatx Immunoelectrophoresis, a powerful modification of Ouchterlony method discussed a bove, i performed using a double diffusion chamber shown in Figure 12.20. The chamber may either purchased from the market or prepared readily by coating a glass microscope slid e with punching appropriate holes in the supporting medium. The antigen in the small round well (1-100 mg). The current is switched on (8 mA, 4-8 volts/ cm] and electxophoresis is allowed to continue for I-2 hours. Immediately after disconne cting the supply, the rectangular well is filled with appropriate antisera and the gel inc ubated temperature in a humid chamber to permit diffusion of antigen and antibody each other. This leads to the formation of precipitin bands at the site lateral to the component has separated during electrophoresis from the rest of the The major advantage of this method is its increased resolving ability due to the of electrophoresis with immune specificity. The technique canbe made quantitativ e has been used to detect particular antigens in sera, tissue or cell extracts, an d culture It has also been used to derermine the purity of a given antigen. Process of tmmunoelectropiwresis: (A] Chamber with pattern cut in agarose/agar. [B} Round wells .filled with antigens: Bovine serum albumin (BSA) and Bovine senan (BS). Eiectro phoress separates the sample nto different oampounds. (C) Addition of antisenun (cubit antt-BS and lea ds to dtffu-sion of antlyens and antibody to Five preclpttln bands. 462 Biophysical Chemistry 7. they are absolutely incapable in.1% to 0. Thus. There are several molecules of DNA i n the individual chromosomes of lower eukaryotes which have a size in excess of 6000 kb. This Is just one example.2%) have been used to resolve extremely larg e DNA molecules. Thus.al ge! electrophoresis techniques. one wants to resolve these large molecules and it is at such a juncture that one painfully re alizes the inadequacy of convention. A solution to this problem was evolved largely due to the efforts of Schwartz an d Cantor in 1984 when they developed pulsed-field gel electrophoresis. puls ed-field gel . The importance of major histocompatibility locus of ma mmals and therefore the need to study it is well known. extrem ely low' concentration of agarose (0. In other words agarose ceases to exercise any molecular sieving effect on these large DNA duplexes. This locus occupies several thousand-kilobas es of DNA Naturally. These large linear duplex DNA molecules migrate through agarose gels at the same rate irresp ective of their size. This behaviour of large DNA molecules is because of a phenomenon known as reptation which means that the molecules migrate 'end-on' through the matrix as if they we re migrating through a sinuous tube. PULSED-FIELD GEL ELECTROPHORESIS Conventional agarose gel electrophoresis can not separate linear double stranded DNA molecules that have a radius of gyration which is larger than the pore size of t he gel. Moreover. As opposed to the con tinuous unidirectional electric fields applied in conventional gel electrophoresis. in order to study this locus. present problems of their own. Secondly they have to b e run very slowly failing which the resolution might be poor. wh enever one has to study a single genetic locus spanning several thousand-kilobases of DNA. These low percentage gels. Why do we need to separate such large DNA molecules? There is a straight forward answer to this question. separating linear DNA molecules whose size is in excess of 750 kb. These large pore gels are capable of sie ving larger size DNA molecules. the molecular sieving being a function of the pore size. Fi rstly they are very fragile and have to be handled extremely carefully. however. it would be desirable if one can separa te it from other genetic material. The problem of separation of large size DNA molecules can be ameliorated to some extent by increasing the pore size of the gel. The original technique of Schwartz and Cantor has now been improved upon so that DNI molecules of size in excess of 5000 kb can be resolved with relative ease. These molecules remain immobile till they reor ient themselves along the direction of the new electric field. (iv) The ratio of the periods of the electric pulses employed to generate the two electric fields. all those molecules of DNA whose reorientation times are le ss than the period of the electric pulse can be fractionated in a size dependent manner. large DNA molecules take a long er time to reorient themselves and are consequently retarded more in the new electric field as compa red to the smaller. (/) The absolute periods of the electric pulses. The Schwartz and Cantor method was capable of separating DNA molecules of up to 2000 kb in size. alternating.electrophoresis uses pulsed. It is here that different DNA mol ecules adapt a behaviour consonant with their respective sizes. which are of extreme importance for determining the limit of resolution of pulsed-field ge l electrophoresla are given below. Thus. (///) The relative field strengths of the two electric fields and the degree o f uniformity of the two electric fields. orthogonal electric fields. Fac tors. . (tO The angles at which the two electric fields are applied to the gel. large DNA molecules become trapped into their reptation tubes every ti me the direction of the electric field is changed. When such a field is applied to a gel. DNA molecules. aL In the apparatus designed by them. The electric fields are continuously switched between . This arrangement produces an electric field which is uni form in both the directions (this is significant since it removes all the problems arising out of nonuniform electric fields in the original apparatus).. This arrangement ensures the migration of the DNA along an absolutely straight track. a vertical gel apparatus with platinum wire electrode position ed on opposite sides of the gel are used. Recent innovations have largely overcome these problems. The apparatus of this type. the DNA path becomes skewed leading to a not-so-good resolution. suffers from certain drawbacks due to which the resolution is not so good. Electroplwresis 463 Instrumentation. The upper limit of separation migh t be significantly higher than 2000 kb. h owever. The pulse in the forward direction is slightl y longer than the one in the reverse direction. aL The apparatus designed by them does not use orthogonal or perpendicular arrangement of electric fields. Instead. For this reason it is also known asfield-inversion gel electrophoresis (FIGE). Another innovation that needs to be discussed is that of Gradiner et. The first innovation is by Carle et. the apparat us utilizes periodic inversion of a single electric field. This innovation (FIGE) makes it relatively easy to resol ve DNA fragments of up to 2000 kb with fairly good resolution.s on the ese DNA horesis.. mbedded . While the ratio between the forward and reverse pulse s is always maintained as a constant. It i s" typical of such apparatus that towards the edge of the gel. This affects the speed and direction of the DNA which depend more and more on the position at which they are loaded into the gel. The original apparatus used alternately pulsed electric fields or perpendicular orientations and linear electrodes. the absolute lengths of the individual pulses might be varied to improve the resolution. The electric eld generated in such apparatus is never uniform.tergents mbedded. Two such innovations of extreme importance are described below. The length of the electric pulses varies depending upon the si ze of the DNA to be separated.the two electrodes and the DNA moves alternately toward one and then toward another electrode in a movement which can at best be described as zigzag. goes a long way in sol ing this The gel matrix.. Thus. 50-60 second pulses are required to a chieve a satisfactory resolution of DNA molecules larger than i000 kb. if tt are extracted form the agarose to submit them to pulsed-field gel electro ' will be broken again. and permeated by an interstitial. There are several companies.g. A new gel. As pulsed-field gel electrophoresis is a relatively new technique. It is therefore quite difficult to isolate long DNA mo lecules from cells. ELECTROPHORESIS ON CELLULAR GELS ] " Long DNA molecules are fragile and molecules longer than I00. phase of easily melted' agarose. no single typ e of apparatus has gained broad popularity till date. such as SOS and proteolytic-enzymes. which are marketing such apparatus.1robp) are broken even by pipettlng. The system is ex tremely useful and DNA molecules of up to 9000 kb length can be separated from e ach other satisfactorily. which have very f ew sit DNA (e. method designed to circumvent this problem is to embed the cells in agarose bloc ks or nicrobeads (diameter 50 mm) and then treating such embedded cells with ion ic d. all the lanes in the gel experience equivalent electric fields. known as the cellular gel.000 bp (0. or 'cellular gel' is a composite containing agarose mirobeads (w ith i point) suspended in. . However. The usual arrangement of th e electrodes is at an angle of 90°. Howev er. Not I). 8. T his condition precludes any horizontal distortion of the resolved DNA bands. while I0 second pulses give a good resolution when the ai m is to separate DNA molecules between 50-500 kb in length. The DNA can be manipulated and cut by restriction enzymes while it i s still e in the agarose. Different laboratories use apparatus of d ifferent designs depending on their needs.. This leads to digestion of cells leaving in tact DNA e in agarose. large fragments to the tune of I mbp will be obtained. the net result of such a zigz ag movement s a straight line and the DNA moves from the well in which it was loaded to the bo ttom of the gel. If one cuts this DNA with restriction enzymes. Significantly. 5' The process of electrophoresis is described in Figure 12. The DNA has now been isolated in the this form it is available for further study. while it is still em bedded in beads can be used as the sample. even after electrophoresis. the DNA extricate themselves from the bead pores and move along on the gel.: hn molten agarose f 1 .21. I I I (% } ' "'" fragments () IntactDNA [ [ . embedded form is used. A. This gel when heated will result in conventional agarose melting off quickly.T. however. The trick is to make whole gel cellular. one could just visuallz zones under ultraviolet light and cut those zone off.. the microbeads. never is the naked DNA handled as such. T..'. Thus.'.. G. It should also be noted that from the time of isolation of DNA from cell to its manipulation for further studies. For further electrophoresis the DNA. When the current is switched on.(B) 3' . C.. It is interesting t o note electrophoresis one handles DNA embedded in the mierobeads and not the naked DNA further manipulation... A..:. will not melt because have a high melting temperature. Beads Molten agarose (c} org CelluFramL gel (G} Broken DNA ... . The mlxture is no w allowed| set. (E) The regon j which the desired DNA fragment has migrated is excised and heated. A.. 3' . (G) Heating up to 55°C releas es DNA ELECTROPHORESIS IN GENETIC ANALYSIS Restriction Mapping Restriction endonucleases are produced by many bacteria and hundreds of differen t of these enzymes have been purified from different organisms.-embedded in * '* unmelted beads Figure 12.21 Ce .. These enzymes have a to cut double stranded DNA in a sequence specific manner. T. Le. 5' . An example is given below. G.. T. any given restric tion will cut the DNA at one and only one base sequence. (D) DNA fragments are now separated electrophoretlcally in th composlte gel .C. A. gels (A) Molten agarose of low melting temperature is with high melting temperatw (55 oc) mkTobea... The rnmre [B] Is then [C] poured tnto an agarose. (F] MId heat (below releases agarose mlcrobeads containing embedded DNA.. and 10D. 39D. 20 25 . However. hundr eds fpieces will be produced by the action of a given restriction enzyme. 35D. their electrophoretic patterns will also differ. This e nzyme cut only at this sequence and will not cut at any other sequence at all. Thus. can therefore differentiate between two different DNA molecules by restriction a nalysis. We subject both these DNA molecules to digestion using the same restriction enzyme. then the fragments produced from molecule X are 5D. will be made clear by the following example. for a large DNA molecule such as that of a bacteria. Likewise the fragments produced from molecule Y are 23D. From the electrophoret ic pattern Figure 12. Different DNAmolecules differ from each other in their sequence. 28D.22 (B) we can infer that the two DNA molecules were different. Since the molecular weight s of the produced from X and Y and even the number of fragments produced from each differs. The seq uence where given restriction enzyme . Figure 12. If we take the molecular weight of both these D NA 100 D. An interes ting possibility here. and since restriction sequence specific cuts we may expect that no two different DNA molecules cut in the same fashion by a restriction enzyme. We subject these restriction digests to electrophore$is. the number and size of DN A the action of any given enzyme will beflngerprint of the DNA molecule. 15D. We are given two DNA molecules having the same molecular weight and want to know they are from the same or different sources.465 X 5 15 35 (B) A restriction enzyme known as XbaI makes a cut in the manner shown above.can cut occurs very rarely along a DNA molecule so tha t if a small from a virus is subjected to the enzyme action we may expect it to be cut in not five pieces.22 (A) shows the position s both of these molecules are cut. 20D. it would be imposs ible. South ern etc. (B) Electrophoretlc pattern of X and Y digested w ith the re-strlctlon enzyme. . process is known as Southern transfer (also called Southern hybridization.of the most useful procedures in molecular biology. Restriction analysis is used for many purposes including identification of delet ion mutaions point mutations in genes.23 39 28 10 I Direction of migration X Y (A) Two DNA molecules X and Y have the same molecular weight but have different sites at which a restriction endonuclease cuts. Edwin Sou thern a procedure. Transfer Suppose that in the above example rather than using a small DNA molecule we woul d used a bacterial DNA and asked to identify a particular gene.) and has become one . so because a large number of fragments would be produced and none will be resolv ed on the gel so that all portions of the gel would seem dotted with DNA. which enables us to identify the fragment containing a particular g ene. and (ii) transferring the denatured DNA from the g el nitrocellulose paper by means of capillary action. The liquid from the tray now rises through the gel .23).466 The principle that is central to this procedure is that a given gene can be loca lized means of its hybridization to a radioactive DNA or RNA molecule ('probe') which has complementary sequence. For paper is placed in atray filled with a suitable buffer and allowed to imbibe the liquid. The whole procedure is descri bed below. the gel is removed and soaked in a solution which is usually NaOH. Agarose gel of DNA Photographic plate Neutralize Nitrocellulose Tissues Filter paper AutoradioRraphy / i i ii Transfer assembly Nitrocellulose Figure 12. Figure 12. Restriction digested DNA is loaded onto an agarose gel (mostly submarine After the dye has run three fourths of the gel.2. A nitrocellulose filter of the same size as the gel is now placed on to p of the gel air bubbles). The gel carefully layered on top of this stack avoiding trapping of air-bubbles between the the gel. All DNA in the gel thus gets converted to single -stranded I This DNA is now transferred to the nitrocellulose paper. However. On top of this is placed again a thick stack of filter papers and these are down by putting a weight on top. DNA separated by electroph0resis is double stranded and again not amen able hybridization. Southern solved these complications by (/) denaturing the DNA wit hin thl gel by treatment with alkali. it is not easy to hybridize DNA while it is in the gel previously this used to be done via the time consuming solution-hybridization Secondly. The paper is now at 80°C so as to fix DNA permanently onto the nitrocellulose. Once the molecules reach nitrocellulose they become adsorbed tightly to the paper and are remaining liquid passes through the paper and is absorbed by the filter papers p laeed at top. The dried paper is n ow inside a fight fitting plastic bag and hybridized with 32p labeled DNA or . The hybridization takes about 16-24 hours. This paper binds both and DNA. The solution has relatively higher ionic strength which hybridization. . The posit ions blackening on the film indicate the location of DNA fragments whose sequences complementary to the sequences of the radioactive probes. Instead is used for the purpose.. RNA does not bind to nitrocellulose in Northern blotting diazobenzyloxymethyl (DBM) paper is used. The paper is then plastic bag and washed in stringent conditions to remove unbound radioactive pro be. paper in then dried and subjected to autoradiography (see Chapter 13).RNA i n a solution volume at 68°C. The name such transfer procedure is Northern blotting. The positions of the DNA molecules adsorbed to the filter are more or less identical to positions they had attained in the gel upon electrophoresis. The first major difference is that RNA is denatured by alkali because it becomes-hydrolysed with such treatment. There are some differences in the between Southern and Northern procedures. Secondly.it the DNA molecules (DNA is not bound in the gel as the gel is mostly liquid). Southern purposes and one of the commonest use is in the screening of c-DNA libraries. The southern transfer procedure has also been extended to RNA now. r-soaked blotting papers. like southern blotting. There are two methods available to transfer proteins to this paper. But this method of transfer suffers from poor yield. Here a sandwich of gel and nitrocellulose is placed in a cassette and is immerse d in buffer between two parallel electrodes When current is switched on. which is quicker and has a better yield of transfer is called el ectroblotting. . Suppose that there is a particular protein whose presence you were interested in determining. the proteins extric ate from the pores of the acrylamide and get deposited on the nitrocellulose retaining the po sition they had achieved in the gel. On top of this another wad of dry blotting papers is kept and the whole arrangement is weighed down by a weight placed on t he top.chosen is nitrocellulose. This process takes just a few hours.468 Western llottln It is by an analogy with Southern blotting that transfer of proteins from a gel onto a nitrocellulose paper has come to be called as Western blotting. is carried ove rnight. through the pores of the gel to the nitrocellulose and beyond into the wad of dry blotting papers. The buffer flows due to capillarity from the trough. To do this the first step is treat the entire paper with a 10 percent bovine serum albumin preparation. The paper with protein s transferred is referred to as a blot. This whole arrangement is transferred into a trough of buffer to prevent drying and t o assure a continuous flow of buffer. The blot made by either of the methods named above can be used for further studi es. The first step in studying proteins fractionated by PAGE is to transfer them on to a paper. The gel on which protein separation has been achieved is placed on a wet block of buffe. Just about 20 per cent of the protei n in the gel gets transferred owing to the small pore size of the acrylamide gel Another method. The first method is absolutely sim ilar to the one for DNA and takes advantage of the capillarity. On top of t he gel is placed the nitrocellulose paper cut to the size of the gel. The proteins trapped in the gel also move along but are arrested by nitrocellulose to which t hey bind irreversibly by hydrophobic interaction. The paper . the same as in Southern blotting. All you have to do now is to probe the blot with an antibody specific for this p rotein. This process. If such is the case. Normally the enzymes us ed in such cases are the horse-radish peroxidase or alkaline phosphatase. Once this incubation is over the specific antibody is incubated with the blot. the blot can be exposed to a photo graphic film overnight and the blackening due to radioactivity will be detectable upon develo ping the film. Supposing the first antibody was raised in a horse. The secondary antibody will bind provided the first one has bound the paper. So that the antibody. The next step is to use the anti-antibody. The antibod y will bind if the corresponding antigen is present on the paper. The blackening will prove that the antigen is present. does not bind throug h non-specific association. the second antibody is an an ti-horse IgG. This is normally an anti-species anti body.Ix. Preferably it has been radiolabeled wit h radioactive iodine .This is necessary because of the tendency of proteins to bind nitrocellulose thr ough hydrophobic interactions. The second antibody is suitably labeled. Oth erwise this too will be washed off. Otherwise it will be washed off. it is necessary that all open sites on nitrocellulose are blocked f irst. which too is a protein. The paper is then . Altenatively. the antibody may be tagged with an enzyme. Second antibodies may also be labeled with fluorescein isothiocyanate in which c ase the presence of an antigen is detectable by fluorescence upon irradiation of the blo t with UV light. Not all bacterial cells will pick up the plasmid.Electrophorests 469 incubated into an insoluble the paper therefore with a substrate solution and the tagged enzyme converts the substrate colour product that is precipitated. An agarose gel i . Sometimes. How do we find out (I) the cells which have picked up a plasmid. There are two qualities in the plasmid that attract biotechnologists to these mo lecules. The cells are made to grow onto an agar medium. they replicate faster and therefore a foreign gene incorporated into them will amplif y much more than it would if incorporated in the chromosome. circular. as stated above. ofa biotechnolog ist consists of purifying a gene of interest and then incorporating it into a suitable plasmid. Plasmids possess a replication origin and therefore replicate independently of the bacterial chromo some. But the genes which confer antib iotic resistance to bacteria do not reside in its chromosome. called the plasmids. As the cells grow. there may be several colonies growing on the petri plate. Detecting Plasmids in Bacterial Cells Antibiotic resistance is a worrying phenomenon. Secondly. The presence of a colour band on is indicative of the presence of the desired antigen. You have to determine which of t hese colonies contains the bacteria carrying the modified/unmodified plasmid. the plasmid replicates and the foreign gene incorporated in it also amplifies. Often the job. gold labeled or even biotlnylated antibodies are used for the purpose of probing.. Some of them may pick up the o riginal plasmid--thaf is the plasmid which has not incorporated the foreign gene. After your gene transfer experiment . Then this modified plasmid is sent inside a bacterial ceil. independently replicating DNA molecules. Often they replicate much faster than the chromosome and therefore one may find bacter ia containing up to even 300 molecules of a given plasmid within a single cell. They are small and therefore one can easily manipulate them. and (2} the cells which have picke d up the plasmid containing the foreign gene? The technique to do so is relatively simple. These genes are present on small. are today used. it is higher in molecul ar weight and therefore migrated slower than the original plasmid. In yet another well you may find a band tat has much less than the plasmid band. the gel is stained with ethi dium bromide. This causes the cens to lyse. The chemical cleavage method is explained below. It therefore does not move. on the other hand can move. however. At the end of electrophoresis. We can now go back to the petri plate. Let us ta ke any . Th e chromosomal DNA is very large and cannot penetrate the gel. This may be the plasmid that has picked-up the foreign gene. Fraction of each of the colonies growing on the petri plate is picked up using a tooth-pick or an inoculation needle and suspended in a drop of a solutio n of sodium dodecyl sulfate. t he cleavage method of Allan Maxam and Walter Gilbert. and the chain terminator meth od . The samples are then electrophoresced. In some wells. a fl)Lcent ban d will be observed right at the origin and will be due to the chromosomal DNA/that hs not rloved. Because it has picked up the foreign gene. This experiment tells us th e colony of has the modified plasmid. pick up this r and grow it separately.s prepared with about 16 wells. This is the band due to the plasmld. The Chemical Cleavage Method : Rather than just discussing this method in a text -book let's try and actually visualize how we sequence a given piece of DNA. In thewells in which the cell-suspension does not contain a plasmid. several methods for DNA sequencing were developed Two of them. Sequencing Just aRer 1975. The plasmid. This is our colony that has the foreign gene. one may observe another band far down in addition to the ba nal at the origin. A fraction from each of such lyse d preparations is loaded onto a well in the agarose gel. 470 Biophysical Ctwmistry arbitrary 10 nucleotide DNA sequence and see how iris manipulated and what the r esullts are.5' 32pTTCAGCCGATOH 3. coli infected with bacteriophage T4. The DNA solution is divided into four aliquots named "G only'. all beginning at 5'-labeled nucleotide and extending up to any one po sition in the DNA strand. Therefore when piper idine is put into this mixture. 'G+A'. To remove this the DNA is treated with alkaline phosphatase which will carryout the following reaction 5' P-TI"CAGCCGAT-OH 3' + H20 . G only In this tube the DNA is incubated with dimethyl sulfate (DMS). Such methylation makes t he glycosidic bond of the methylated G residue susceptible to hydrolysis. In most cases the DNA fragment will have a phosphate at the 5'-end. Things will become clearer later in the discussion. the DNA is incubated with [-32p]ATP and the enzyme polynucleo tlde kinase from E. and 'C+T'. (If you do not understand how this is done or what exactly the above means. 5' P-'ITCAGCCGAT-OH 3' The first step in the chemical cleavage method is to radioactively label one end of the single stranded DNA. the DNA gets broken at those positions where the G residues h . The following reaction Will take place 5' OH-TI'CAGCCGAT-OH 3. This compound has a property of methylating only G residues at position N7. don't lose hope.) Let's see exactl y how this is done. Let's see whathappens in each tube. This is done in a manner that each DNA molec ule is broken at an average of one randomly located susceptible bond. The basic strategy of the chemical cleavage method is to specifically cleave the end-labeled DNA at only one type of nucleotide. +ADP [7-32p]ATP The DNA now carries a label at its 5'-end. 1. +A-p-p-32p-------.5' OH-TI'CAGCCGAT-OH 3' + PI Once this is done. This ensures production of radioactive fragments. Following is the sequence that we randorrfly select. 'C only'. In some of the strands the 5 G will rands it will be the 8. in some strands both theG residues will be methylated. Very small concentration of DMS ts used. In another str and some other G will become methylated. When piperidlne is mixed then. Now let's see the trick here.ave become methylated. The process is entirely random. one at position 5 om the labeled end. Such a treatment will result then in DNA strands of varying length depending on which G has become methylated in a gi ven strand. One strand will be 4 nucleotides long and the o ther will be 7 nucleotides long. However. There are two G and the other at position 8 counting fr become methylated and in some of the st the following fragments will be created 5' 32P-TrCA. Concentra tion so small that it cannot methylate every G of evenj DNA strand. 5'P-TrCAGCCGAT-OH 3' . To be sure let's see which fragments will result if we give the above treatment to our chosen DNA strand. This ensures that in one D NA strand a particular G is methylated while all other G residues are spared. it is easy to see that in such strands the piperidine treatment will create a 4 nucleotide lon g fragment. and 5' 32P-TTCAGCC. . The original sequence of our strand is : residues present here. . Of course. But these strands will be there in all t ubes. The polyacrylamide percentage is very high ma king the pore size extremely small. TrCA. it is run at 70° C . C.. Tr. C+T IfDNA is reacted with hydrazine (NH -. Five labeled fragments. C. G. The four different ly fragmented samples of the DNA. with an aci the glycosi Again the s G residues TIC. A+G. and 9 nucleotides in length. It also contains 8 M urea. and 6 nucleotides long. Of course.5M NaCI.Electrophorests 471 9. TI'. (2) the DNA fragments will migrate as straight rods. 3. T. the following fragments will be created. TI'CAGC. Following fragments will be created. Four fragments. and 8 nucleotides in length. C+T) on a sequencing gel. therefore. are simultaneously electrophoresed in pa rallel lanes (labeled G. A+G. 2. 5. only C residues react well. Three fragments. A+G Here the DNA is protonated rather than methylated. 4. TFCAG. Together. measuring 1. The following piperidine treatment will therefore destroy glycosidic bonds befor e only C. The sequencing gel is a long (up t o 100 cm). 7. and C+T. measuri ng 3. In this tube. thin (just 0. 5. TTCAGC. C only If hydrazine treatment is carried out in presence of 1. TTCAG. The following fragments will be created in this tube. TrCAGCC. TTCAGCCG. both A and G residues are protonated at equal rates.. 6. in some strands no base will be destroyed and therefore a whole labeled piece will also be there.NH ) and this is followed with piperidine treatment. the glycosidic bonds before both C and T are destroyed. If DNA is treated d. 4. all labeled at the 5'-end. TI'CAGCCGA. Protonation also makes dic bond susceptible and therefore liable to break upon piperidine treatment: trategy is the same as in 'G only' : protonation of preferably only one of the A or should take place per strand. and (3) the DNA fragments will separate . Moreover.. these three conditions ensure that (1) no hydrogen bonds can form between nucleotides. The next step is probably the most important for sequencin4.1 mm) polyacrylamide slab. 2. the high polyacrylamide percentage ensures that they migrate as separate bands. If it appears in both the lanes. The sequence of the DNA may be simply read off this autorad/ograrn.24). the sequencing gel is exposed to a photographic fi lm. it means a G. Let's try to picturize how this autoradiogram will look like for the fragments t hat we have created of our chosen DNA sequence (Figure 12. No w read. it is read as a C. it means a T. Now just start reading the sequence from the smallest fragment onwards. Even if two fragments differ in size by as littleas one nucleotid e. the photographic film will be blackened at positi ons where the fragments have migrated to. When a band appears only in the C+T lane and not in the C lane. from the bottom. If the band appears in both the lanes. Likewise when a band appears only in A+G and not in G. that is. it means an A. Once electrophoresis is over. The sequence that you can read is . Since the fragments are radioactive.only according to their size. The technique that allows us to delineate these protein binding sequences is called 'DNA footprinting'. Th basis of the technique is surprisingly simple. What about the 5'-nucleotide? How do we read it? Remember that we took only one strand for fragmentation. and develop it like above. DNA Footprlnting There are proteins which bind to specific sequences within the DNA. Of course. Sequence has to be read by the presence of bands in the two lanes. up to 300 bases can be read on a single gel. This is a norm. or even inhibit it. you would be able to read the 5'-nucleotide. presence in both lanes means a 'C'. It is very necessary that we find exactly what these sequences are if we have to understand the molecular processe s underlying gene expression. Nor mally. Transcription factors are such proteins. w hat is just one nucleotide if you have been able to read 300. 10 nucleotide sequence. By such bind ing they regulate the expression of genes .24. Schematic autoradiogram of the sample DNA fragments as they migrat ed on the sequencing gel on the basis of their size. Likewise a band present in the C+T lane without a corresponding ban d in the C lane means a "F. A piece of DNA is incubated wi . In fact this technique is just a variation of the seq uencing technique dscribed above. T he 5'-nucleotide cannot be read in the chemical cleavage technique.472 5'-TCAGCCGAT-3' Figure 12. you have the option of ignoring to read that nucleotide. After all. For the sake of understanding we have taken a small. A band present in the A+G lane without a crresponding band in the G lane means an 'A': presence in both lanes means a 'G'. DNA is double stranded and the two strands are anti-paralleL Thus if you take the other strand now. Sequence has to be read bottom u pwards. Moreover all enzyme s concerned with DNA synthesis or DNA directed transcription also bind DNA. If you compare this with the sample sequence you will find that the two are virt ually the same except that the first nucleotide (a T) has not been read.they can make the transcription of the concer ned gene faster. If the digested DNAis now electrophoresed.th a nonspecific DNase. The conditions are manipulated such that the DNase can make just one random cut per piece. This situation is reminiscent of the situation that was created i n the sequencing experiment. the fragments will migrat e according to their size if the gel has been prepared just as described for the sequencing exp eriment. . 25). translation. and NMR. need expert analy sis. DNA footpHntng. and require large amounts of material and quite expensive instrumentation. are due to specific interaction between nucleic acids and proteins. no elaborate instrumentation and provides very good results about nuclei c acld-protein interaction which can be interpreted with comparative ease. then the protein will give protection to that sequence to which it is binding. Gel retar dation or band shift assay on the other hand. Gel Retardation or Band-Shift Assay Studying nucleic acids-protein interactions is of fundamental importance in unde rstanding the cell growth and behaviour. there is a gap n the ladder of segments produced. transcription. Thus when DNA Is fragmented and mjrated on electrophoretlc gel. Such basic elementary processes as DNA replicatio n.25. all these techniques are tedious. If now in the solution the concerned protein is present then it would bind to that sequence. The rest of the DNA will be cut just as before.473 DNA 5 10 15 20 25 Figure 12. wewill be able to tell the exact sequenc e of the protein binding region. However. Nuclease cannot digest that portlon of DNA to whic h a protetn is binding. Comparing of :he previous gel where protein was not present and the gel where the protein is present tells us the region where the protein is binding. The gap corresponds to that sequence where the protein was bound. These interactions have been studied using X-ray crystallogr aphy. controls of gene expression etc. If now a DNase digestion is resorted to. Although this . Now if the resulting fragments are electr ophoresed again. Due to its simplicit y. is a technique which requires quite small am ounts of sample. this technique has become the most widely used for studying protein nucleic acid interactions. Consider that this piece has a protein binding sequence. circular dichroism. If we seque nce the entire piece with chemical cleavage technique. there will be a gap in the ladder : the fragments for the protected region will not be there (Figure 12. Linear DNA molecules migrate on an electrophoretic gel as a monotonic function o f their length.protein interactions. If before migrating on the gel. in most of our following discussion we would limit ourselves to DNA.technique has been used to study both DNA and RNA interactions with proteins. rather in a worm like fashion known as reptation. DNA fragments are incubated with a protein. a co mplex might be . which interacts with nucleic acids. c-AM P) must be added to the incubation mixture. Since the complex formation is mostly a result of ionic interactions. (D) and (E) involve bin ding of DNA to either a . however. Many proteins need cofactors ( e.26) Figure 12. and/or (ii) the charge of DNA . non-speclfic binding can also take place. e. Thi s retardation of nucleic acid mobility might be because (i)the mass of the DNA-protein complex is certainly higher than that of the naked DNA leading to a lower mobility.. If the protein is a DNA binding protein. This complex would then migrate much more slowly than the DNA fragment w ould have migrated alone {naked).g. This is the principle of gel retardation assay. H owever. it is important to use buffers which are of low io nic strength and thus do not interfere in c0mplexation. It. Situans (C). S (A) and (B) correspond to bldng of multlple copes of a protein and thus the retardation is higher. Apart from these intrinsic fa ctors.quite simple. seems that it is mostly the protein mass that influences the mobility of the com plexed DNA (see Figure 12. and the temperature ofelectrophoresis can also affect the mobility of complex or naked D IA. The technique itself is . under these conditions it will complex with a given DNA sequence for which it is specific. the composition of b uffer. The llrst step is to add the protein in w hich one is Interested to a solution of linear double stranded DNA fragments and to incubate under suitable conditions to promote complex formation.. the rrrigratlon of DNA ts is largely dependent upon the mass of the proteOl wffh which they complexed.fragment has been modified due to ionic interaction between itsel f and the protein.474 Biophysical Chemistry formed.26 Effect ofproteln mass on migration of DNAfragments. If proteln indu ced changes tn DNA ¢onfommtWn oncl charge are ignored. These non-specific interactions can be avoided by ad ding salt to the binding mixture so that non-specific ionic interactions will . other external factors such as the composition of the gel matrix. and/or (iii) the interaction with protein has induced certain conformat ional changes in the DNA which impede its movement through the gel.sgle copy or to truncated proten (truncated circles) and the retardan is corre spondly lower. retardation. Another way is to add excess of synthetic DNA copolymers such as poly-dA-dT. or poly-dl-dC etc. Normally slab gel electrophoresis with polyacrylamide gels is routinely used. Alongside t hese lanes. After the binding incubation is over the mixture is loaded onto the lanes i n the gel.be disturbed. naked . and stoichiometry of DNA-protein in teraction. co-operativity.Electrophoresis 475 DNA fragments are also loaded to run as standard. This visualization consists of autoradiography. visualization is carried out. . Standard electrophoresis buffers such as the THE (90 mM Tris-borate. 2 mM EDT. it 2 nd DIMENSION O r I t E a N r . In the lane where naked uncomplexed DNA is migrating. The complex can then be extracted from the gel and further studies can be performed. I mM EDTA pH 7. A comparison between the lanes in which the DNA-protein mixture wa s loaded and the lane in which the naked DNA was run aids in identification of the DNA fa gment which was retarded and therefore the fragment which interacted with the given protein. After the electrophoresis is over. The technique has been us ed to determine binding constants. those DNA fragmen ts which became bound to the protein will migrate at a reduced rate (retarded)while the u ncomplexed DNA fragments will migrate depending on their mass. In other lanes.w here complex forming incubation was carried out with addition of a protein. Due to the simplicity of the technique and due to the quality of results obtaine d using it routinely. this technique has found many applications. or it involves ethidium bromide staining and visualization under UV light if unlabeled DNA was used.9) are also use d routinely. the migration o f the individual fragments will be strictly a function of their mass.3) or the TASTE (40 mM Tris-acetate. however. Electrophoresis is carried out using low ionic strength buffers. if labeled DNA was used. pH 8. s d I a 0 t N i 0 12.27 Two-dlmensl gel electrophoresls for genorne scanning. Gelretardan of res trtlon dest of the genome is performed in the jqlst dimension. The pmteln-DNA complex s denatured o n the gel by heating or by treating the gel with some denatwunts. Electrophoresls is now perf ormed under denaturlng conditions in the second dimension 90o to the flrst. A retarded DNA fragm ent (Heavy line), which could not be detected tn the first dimension, runs ahead of th diagonal in the s econd dimension because it runs according to its octual sze. This leads to its identtJkUn. Arrow ndUes the frag- 476 BiophysaI Chemistry has been used to define binding sites, binding requirements, protein domains inv olved in interaction and in genome scanning. The last named application, v/z., gen0me scanning is extremely important. Gel re tardation technique is quite sensitive so that it can select rare molecules from a large p opulation. This ability of the technique has been used to scan specific binding sites withi n whole genomes. The genome is fragmered and incubated with a protein(s). A problem, however, may frequently arise here. Due to the complexity and abundance of DNA fragments the retarded DN A-protein complex might co-migrate with larger uncomplexed DNA fragments. It would then be very difficult to resolve the DNA-protein complex from such gels. A two dimensional m ethod has been evolved for precisely such situations and this method allows one to resolve the DNA fragment involved in protein binding very easily. The DNA-protein mixture is rtln on the gel in the same manner as described above (Figure 12.27). The gel is now treated to denature the DNA-protein complexes either by heating or by incubating with denaturing agents. Electrophor esis is performed again in a second dimension, 900 to the first run. The protein'DNA bin ding has been disrupted and the DNA fragment involved in binding now migrates according to its real size, ahead of the diagonal (Figure 12.27). This technique has been used to localize p rotein-blnding sites in/ambda and Mu genomes and even E-colt chromosome. Suggestions For Further Read/rig Moving Boundary Electrophoresis I. Moore, D. H., Electrophoresls in Physical Techniques in Biological Resea rch (D.H. Moore, ed.) Academic Press, New York, 1968. Paper Eleetrophorsis 1. Peters, H., Paper Electrophoresis: Prlncples and Techniques in Advances in Clinical Research, Vol. 2, 1959. Gel Electrophoresis and Other Techniques I. Charmbach, A., and Rodbard, D., Po/yacry GeIElectrophoress, Science, 172 :440-451, (1971). 2. Gordon, A. H., Electrophoresls of Proteins in Polyacrylamkie and Starch Gels in Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 1, (T.S. Work and E. Work , eds.) North-Holland Publishing Company, 1974. 3. Gould, H. and Matthews, H.R., Separation Methods for Nucleic Acids and O llgonucleotides in Laboratory Techniques in Biochemtry and Molecular Biology, Vol. 4 (T.S. Work and E. Work, eds.) North-Holland, 1976. 4. Gabriel, 0., Analytkml Disc GeI Electrophoreses in Methods in Enzymology , Vol.22, (W.B. Jakoby, ed.) Academic Press, New York, 1971, pp. 565-577. 5. Weber, K., Prlngle, J.R., and Osborn, M., Measurement of Molecular Weigh ts of SDS-Acrylamlde Gels in Methods in Enzymology, Vol. 26 C, (C.H.W. Hits and S.N. Tlmasheff, eds.) Academic Press, New York, 1972, pp. 3-27. 6. Gaal, O., Medgyesi, G.A. and Vereczkey, Electrophoresls in the Separatio n of Biological Macromolecules. Wiley, Chichester, 1980. 7. Catsimpoolas, N. and Drysdale, S, (eds.), Biological and Biomedh::d Appl ications oflsoelectrlc Focussing, Plenum, New York, 1977. 8. Vesterberg, O., Isoelectr/c Focuss/ng of Prote/ns, in Methods in Enzymol ogy, Vol. 22 (W.B. Jakoby, ed.).Academic Press, New York, 1971, pp. 389-411. 9. Sambrook, Fritsch, Maniatls, Molecular Cloning: A Laboratory Manual, Col d Spring Harbour Laboratory, 1988, pp. 650-652. 477 I0. Dear, P.H. and Cook, P.R., Cellular gels: Purifying and Mapping Long DNA Molecules, Biochemical Journal 1991, 273: 695-699. If. Lane, D., et. aL, Use of Gel Retardation AnalySis, Microbiological Revie ws, Vol. 56, 1992, pp. 509-524. ' Exercise I. What will be the relative electrophoretic mobilities of alanine (pl = 6. 02), arginine (pl = I0.76), glumatic acid (pl = 3.22), serine (pl = 5.68), and tryptophan (pI = 5.88) at pH 5.68 ? 2. Isoelectric pH of a few proteins are given below: Protein Isoelectric pH Egg albumin 4.6 Serum Albumin 4.9 Urease 5.0 -lactoglobulin 5.2 Hemoglobin 6.8 Myoglobin 7.0 Chymotrypsinogen 9.5 Ribonulease 9.6 Cytochrome C 10.6 Lysozyme 11.0 At what pH would you carry out electrophoresis for the most effective separation of the following protein mixtures ? (a] Egg albumin and hemoglobin (b] Hemoglobin and ribonuclease [c) Egg albumin, serum albumin and urease. Sickle cell hemoglobin migrates at a different rate than normal hemoglobin. You are given purified normal and sickle hemoglobin as standards. You have isolated hemoglobin from 15 patients suffering from anemia. By comparing the electrophoretic mobllities you want to find out which of these are sickle hemoglobin. Which mode of gel electrophoresis will you use? E. Co//genome was isolated and a small DNA segment of it was arr/plifled a milli on folds with the help of suitable primers using PCR technique. When the whole mixture was electrophoresced on a 2 per cent agarose gel and stained with ethidium bromide, only one band corresponding to the size of the amplified segment was visible under UV lig ht. If the same mixture was electrophoresced on a standard polyacrylamide gel, several band s were made visible by ethidium bromide. Give explanation for these observations. A researcher is carrying out slab gel electrophoresis of 24 DNA samples. He has completed the assembly, poured the buffer which has a pH of 9.0 and has loaded the samples . When he switches on the current he observes more bubbles coming out from the lower buffe r reservoir as compared to the upper reservoir. Is he in trouble? Aliquots from the same protein-containlng sample were subjected to SDS-PAGE and cation exchange chromatography. After staining SDS-PAGE reveals Just one sharp band whi le one obtains two distinct peaks at different elution volumes in cation exchange chrom atography. What are your onclusions about the composition of the protein solution? 478 7. BWphys X-ray crystallographic studies of a given protein have conclusively proved that the consists of four peptlde Chains in association with each other. Yet SDS-PAGE of the protein gives just one band. How many explanations can you provide to jus tify above observation? 8. You have a mixture containing two components. Given the choice of varyin g either pH, strength, or temperature of the medium, which condition will you choose to vary separation? Why? Why will you not easily choose to vary the other two conditions ? 9. Ybu are given a mixture containing two proteins having similar molecular weights, and Are you confident that they will migrate as a single band? I0. You have migrated a given mixture of proteins on two gels of same compos ition using of different pH. On each gel you get two bands. Wig you be sure that proteins? 11. Why is electrophoresis done in solutions having low salt concentrations? 12. You were given a piece of DNA. When you electrophoresed it on polyacryla mide, it gave you single band. You subjected this DNA to digestion with a restriction enzyme. Post you still get a single band upon electrophoresis. In how many ways can you justi fy result? 13. In the above experiment, if it was told to you that the given piece of D NA contained a site for the given restriction enzyme, which of the above conclusions would 13 .ISOTOPES IN BIOLOGY The phenomenon of natural radioactivity was accidentally discovered by the Frenc h Henri Becquerel when he found that crystals of potassium uranylsulphate emitted a radiation, which could blacken a photographic plate. Subsequently r radioactive elements like radium, thorium, and radon were discovered. Of these , has become linked with the name of the legendary Madame Curie, whom we owe much understanding of the phenomenon of radioactivity. For obvious reasons, the aboe-described natural radioisotopes have a very limite d use in '. The biological applications of radioactivity had, therefore, to wait for the development of instruments as the cyclotron and the nuclear reactor that made possible producti on of radioactive materials. Using the nuclear reactor it became possible to produce for those elements, which are commonplace in biology, for example, carbon, sulphur, hydrogen etc. Since then the use of radioactive isotopes in biology has legion, so much so that it is difficult today to imagine of an experimehtal setu p without However, before describing their use and the instrument involved, it would be pr oper consider the structure of the atom and ask why some atoms are radioactive. An atom is composed of a positively charged nucleus around which the electrons s ettle , the charged clouds. The mass and the stability of the atom reside predominantl y in the Atomic nuclei are composed of two major components; protons which are positively and neutrons which are neutral. The number of electrons (negatively charged) n a n is always equal to the number of protons in the nucleus thereby making the atom neutral. This number is known as the atomic number (Z). Neutrons are uncharged equal to that of a proton. The sum of protons and neutrons given nucleus is known as the mass number (A). Thus, A = Z + N. Where N denotes the B an ItoI? The word isotope is derived from the Greek /so (same) and topos (place) and thus occupying the same position in the periodic table. It was the chemist Soddy in 1 913 first established the existence of atoms of the same element with different atom ic weights cdled them isotopes. Isotopes are atoms of a given element, with identical numbe r of protons nucleus, the same pattern of electrons in the clouds around the nucleus and ther efore the ', characteristics, but with different number of neutrons and consequently dijer ent The number of stable isotopes that elements possess varies widely, for example, possesses six, carbon has three, while sodium has only one. There is a conventional form of describing isotopes The superscript to the left of the symbol indicates the mass number A, while the subscript to the left indicates th e number Z. For example, Na represents the sodium isotope, atomic number 11 and lgure 13.1 480 Bioptujsical Chemis mass number 23. Since the atomic number of any given isotope of an element is th e same, th subscript is often omitted. The sodium isotope may then be described merely as 2 3Na. What is Radioactivity ? In general the stability of an isotope of a given element is dictated by the rat io of neutron to protons in the nucleus. The relationship between the number of protons and ne utrons in nucleus can be seen by plotting the values of N against Z for known isotopes (Fi gure 13. I). ] will be evident from the graph that stable isotopes for elements with low atomic numbers ten, to have an equal number of neutrons and protons, whereas stability of elements w ith high atomic' numbers is usually associated with a neutron to proton ratio in excess o f one. It thus be surmised that N : Z ratio of a stable isotope lies within narr ow limits so that an isoto outside these limits will be unstable. Such a nucleus will try to adjust its N : Z ratio toward stability, giving out radiation in the process. Thus the phenomenon of radioacti vity can be see as an attempt on the part of the isotope to achieve stability. The first discovered natural radioactive elements were among the heaviest elemen ts. Th is logical, since only these elements could have existed for a length of time as long as the age the earth. With the advent of the nuclear reactors, however, it became possible for tl scientists to bombard atoms with neutrons and alter their N : Z ratio, thereby p roduci artificial radioisotopes. 120 II0 I00 90 80 70: O 50 10 .::¢. . :-" .#. I0' 201 30 40 50 60 70 80. 90 100 Number of protons (Z) Plot showing relationship between the atomic number (Z) and the number of neutro ns (N) in nucleus. The solid llne represents a neutron/proton ratio of unlty. Black dots r epresent isotopes are stable. Isotopes falllng away frorn the dotted zone would be unstable. RADIOACTIVE DECAY It is clear from the above text that the ratio of protons to neutrons is of impo rtance detenTdning whether or not an element is radioactive. An unstable radioactive nu cleus " Isotopes in Biology 481, reach or approach stability through emission of radiation' This emission of radi ation is known as rad/oact/ve decay. A radioactive compound may decay by any one or more of the several ways described below. t Ngatron ¢mulion. In this case a neutron is converted to a proton resulting in th e ejection of a negatively charged beta (() particle known as a negatron (-we). NEUTRON PROTON + NEGATRON A negatron is an electron, The term negatron is, however, preferred because it e mphasizes the nuclear origin of the particle. The negatron emission results in loss of a n eutron and gain of a proton. Consequently the N : Z ratio decreases while the atomic number (Z) inc reases by one. The mass number (A) remains unchanged. The isotope thus becomes a new element. 3 2p can be cited as an isotope frequently used in biology which decays by negatron emission . 32 P-"' 32 S ÷ we 15 16 (iO Positron emi,ion, The positron is understood to be a positively charged part icle. Isotopes like 22Na and C decay by emitting positrons, During positron emis sion a proton is converted to a neutron. PROTON NEUTRON + POSITRON Positrons have a transient life and are annihilated on .coming in contact with e lectrons. The mass and energy of the two particles is then converted to two gamma rays emi tted at 180o Positron emission results in loss of a proton and gain of a neutron. Consequentl y, the : Z ratio increases, the atomic number decreases by one, while the mass number r emains example of positron emission is given below: t||OAIpha emiion. The alpha particles are heavier than other emitted particles b ecause consist of two neutrons and two protons. Alpha emission results in a decrease in atomic number of two and a decrease in mass number of four. Decay of 2U is cited as an example. 22U--- 234 Th + 4 He 90 2 (iv) Electron cttpture or EC-decay. This is similar to positron emission in that a proton is This, however, is accompanied when an orbital electron is captured the nucleus. The capture changes the pattern of orbital electrons. The remaining electrons themselves emitting gamma .ray or an X-ray in the process. 4. 4 + y/. a:'--ra-''-r^'251vln÷e ------> 24Jr (v) EroSion of gamma ra. y-rays are usually emitted when the nucleus of an atom is This frequently accompanies alpha or beta particle emission. Gamma emission not lead to a change, in atomic or mass numbers. In addition to the patterns" of decay stated above, there are other patterns of radioactive which are of no practical interest to a biochemist. Some examples of such patter ns are transition and sontaneous fission. 482 Biophyslcol Chemistry Radioactive Decay Energy The principle unit of energy in nuclear physics is electron volt (eV): This is t he energy acquired by an electron when it is accelerated through a potential difference of one volt and is equivalent to 1.6 x 10-9 J. It is a very small amount of energy and usually KeV (thousands of electron volts) and MeV (millions of electron volts) are used. Isotopes emitting a-partlcles are most energetic, their energies falling in the range of 4.0-8.0 MeV, whilst - and y-emitters generally possess decay energies of less than 3.0 MeV. Rate of Radioactive Decay The disintegration of an unstable nucleus is an event so completely unpredictabl e and random that the only thing which can be said is that an unstable nucleus will de cay within a given period of time. For a particular isotope, the proportlon of nuclei that decay in a given time is a constant known as its disintegration constant. If the decline in activ ity of a radioactive isotope is plotted against time (Figure 13.2)'a tpical exponential form is obtai ned. II0- TOO . 701 2 3. 4 5 6 Time in half-I/fe Figure 13.2 Radioactive decay. The units of tlme are in half-llves. This may be mathematically be expressed as a simple first order process -dN - N dt -dN where --di-- = the number of atoms decaying per small increment of time, N = = a The The the total number of radioactive atoms present at any given time, decay constant, characteristic for a given isotope, and t =time. negative sign is essential because the activity is decreasing. above equation may be rearranged to get a term for . -dN , N or k = -dN or = -dN/N dt N.dt dt Isotopes in Biology 483 The above equation means that k is that fraction of radioactive atoms which deca ys per unit time. We may integrate the above differential equation -dN dN -- .IN, o dt N Carrying out integration between limits of NO (radioactive atoms existing origin ally) and N {radioactive atoms remaining after time t) and between the limits of zero time ( t = 0} and any other time, t, we get ,!' d- -k S dtorln= N m = -kt No or InN° = tt or 2.3031ogD NO = .t N N or N =N e-U or IogN=- t+IogNO 0 2.3.3 Another constant for any given radioisotope is the time that is required for the original to fall by a half. This is referred to as the half-life and is used more commonly than the constant because it is of much more practical utility. The half-life, tI /2, of a given and its decay constant, , may be tied in a relationship as follows or or 2.3031ogm= N° Xt or 2.3031og---1 = kt N 0.5 2.303 log 2 = kt or 2.303 (0.301} = t 0.693 kt 0.693 0.693 .'. t2 = orkffi t2 The above relationships allow us to ca/culate half-life of a given isotope if it s decay constant krown and vice versa. The curve in Figure 13.2 may be converted to a straight line by plotting time ag ainst log N. this curve touches the y-axis is,log NO and the slope is -/2.303. Half-life is a widely varying property. For example, the half-life of 14C is 500 0 years, whereas has a half-life of 15 hours only, When several different isotopes of the same el ement are for experimental use, the half-life is one of the most important factors in tell ing us to use. 484 Units of Radioactivity The unit of radioactivit is the cur/e (Ci). It is defined as the quantity of rad ioactive material in which the number of nuclear disintegrations per second is the same as that in I g of radium, i.e, 3.7 x 10l° (3.7 x 101°s-). The curie is a large unit and only millicuries (mCi) are needed for tracer applications. The curie refers to the number of disintegrations actually occurring in a sample rather than the number of disintegrations counted in a radiation counter. Normally in radiotracer experiments the radioactive isotope is added mixed up wi th the stable isotope. It therefore becomes necessary to express the quantity of radioi sotope present per unit mass. This is known as the specific acttv/ty and may be expressed as (0 disintegration rate (ds- or d min-), ( count rate (ct s- or ct min-), or (/t0 curies (mCi or Ci ) per unit mass of the mixture (Ci or mCi/mole or gram). The international system of units (SI) has specified the term bequerel (Bq) to r eplace the term curie, which is currently in use. The bequerel is defined as one disintegra tion per second. A curie is therefore equal to 3.7 × 10° Bq. PRODUCTION OF ISOTOPES Bombarding the target nuclei with alpha-particles from naturally radioactive sou rces was the way in which artificial isotopes were produced initially. An example is affo rded by the work of Cockroft, and Walton. In 1931, these workers bombarded lithium target nuclei with hydrogen nuclei and found that many such collisions, resulted in the emission of high ene rgy alphaparticles: Li+ H----- 2 [ He] In the above example, the lithium nuclei and the proton have interacted to produ ce a pair of helium nuclei. The cyclotron, an instrument that accelerates protons and other charged particle s to very high energies, was developed in the late 1930s. Although this development made a large number of nuclear transformations possible, perhaps the most important development was the advent of nuclear reactors. The significance of this development was that the scientist s could now bombard the target nuclei with uncharged particles like the neutron. Since it do es not bear a charge, it is easier for a neutron to interact with the target nuclei without ge tting repelled. After this development, the use of isotopes in all branches of industry and scie nce became legion. Most of the isotopes used in biology are today produced by neutron bomba rdment. An example of the kind of interaction that takes place in a nuclear reactor is give n below: :271 + on -- 2sI + Y No chemical change in the target nucleus is involved in the above reaction. It i s therefore almost impossible to separate the radioactive isotope (lsI} from the stable isot ope (127I). There are, however, other reactions where the target is changed chemically. One such e xample is given below: 35 I 35 17CI +0n--- 16S÷ p The radioactive isotope here is chemically distinct from the target stable isoto pe; the two can be separated easily. SYNTHESIS OF LABELED COMPOUNDS A prerequisite for these isotopes to be used in biological studies is their inco rporation in the bio-organic compound which is to be studied. For example, 4C has to be incor porated into glucose if we want to study the fate of the glucose in the body..The glucose mol ecule which has one or more of its carbon atoms (2C) replaced by (4C) is known as a labeled mole cule (glucose), while 4C is the/abe/. It is also necessary that the label should be incorporated in the position, which is appropriate for the experiment being carried out. Thus, out of the six carbon atoms that glucose contains, the label should not get incorporated at any position fr0 .m 1-6 at random. The labeling procedure should be such that the experimenter should be able to co ntrol the position being labeled. Two labeling procedures are widely used to prepare such labeled compounds. They are discussed below. Organic Synthesis The synthetic methods of organ/c chew/st are being used on an ever-increasing sc ale to prepare compounds with the isotopic label in defln/te positions in the molecule. The synthetic method used for labeling is usually an ordinary one. This method, however, has t o be mod/fled /n such a way that the y/eld of the labeled compound is the highest and the isot ope, wh/ch is the most costly mater/al in the reaction, is conserved. The preparation of acetic ac id labeled with 'C in the carboxyl group is.cited as an example. The reaction consists of treatm ent of Grignard reagent from methyl bromide with 4CO2. CH3MgBr + 4CO2 CH3,:'COOMgBr + H20 ----->CH.:'COOH + Mg(OH)Br The reaction is actuaLhf earned out using Bat'COs which when subjected to the ex perLmenta] set up releases CO2. This gas is then del/vered to the container of Grignard rea gent. 486 Biophystcal Chemistr9 The above example is very simple where the method of conventional organic synthe sis did not need to be modified much. There are, however, other labeled syntheses where the organic synthesis method has to be modified profoundly. One such example is the synthesi s of DLlysine labeled in the z-carbon atom. One interesting problem arises here. Since this synthesis is a multistep process, the isotope can be introduced either at an early step in th e experiment, or in a later step. The latter option seems rosy from the point of view of conservi ng the radioactive isotope. However, it is experimentally found that introduction of isotope at a l ater step in the experiment does not yield the desired product. Therefore, even though the yield would be less, it is necessary that the isotope be introduced at an early step in the synthesis . The reaction steps are givenbelow. (0 4CO2 + 4K + NH3 -----> KH + 2KOH + K4CN (//) KCN + Br(CH2}3CI CI(CH}4CN + KBr /COOCH. /'COOCH (I) N'C'CH.CH'CH C] + NaCH ----> N'4C.CH2-CH'CH'C'H + NaCl cooc,H cooc H dlomalonie ester /COOCH N*C.CH.CH.CH.CH .+ CH--O--NO N*C.CH.CH.CH..COOC2H COOCH Ethyl nitrite NOH Reduction (v) NC.CHa.CH.CH..COOCH5 + 8H -> HN-CH.CH.CH.CH.CH.COOCH + I-IO Noa Lysine ethyl ester Lysine was finally isolated as lysine hydrochloride. The yield of this reaction is fairly good. Several labeled compounds are synthes/zed by way of organic syntheses. Biosynthesis Labeled compounds with increasing complexity, which cannot be synthesized by the laboratory, are made through biosynthesis. Example can be cited of sugars, gluco se, fructose, sucrose and many others. These labeled sugars can be produced by photosynthesis under controlled conditions, with 4COin the gas phase. This biosynthetic method gives high yields of uniformly labeled sugar molecu!es. The sugars can then be extracted, separated b y chromatography and, crystallized by mixing with a carrier compound. Loss of the isotope is minimal in biosynthesis. Biosynthesis has been employed to prepare such labeled pharmacodynamic agents as digitoxin, morphine, and nicotine. Usually microorganisms are used in such label ed biosyntheses. However, ff synthesis of a metabolite peculiar to a particular spe cie is required, the specie concerned might be used for such labeled synthesis. Other examples of biosynthesis oflabeled compounds are (0 plant pigments such as chlorophyll, leaf carotenoids, (/0 alkaloids, (rio labeled cellulose and glycogen, (iv) haemoglobin with the help of radioacti ve iron, (v) thyroxine by tntroductlon of radioactive ''I iodine and (vi) cholesterol by injecting .labeled acetic acid, The overriding concern during biosynthetic labeling is of assuring the purity of the desired compound, This is necessary so that erroneous results due to radiochemic al contamination can be avoided. Isotopes in Bology 487 The nomenclature of the labeled compounds is based on the Geneva system. Thus, a n acetic acid molecule labeled by 4C in the carboxyl group (CH3.14COOH) is indicat ed as 1-C-14 acetic acid. If the isotope used for labeling was 3C, the labeled acetic acid would be known as I-C-13 acetic acid. If the acetic acid is labeled tn the methyl group w ith aC, the nomenclature would be 2-C- 14 acetic acid. Similarly, ATP labeled in they-phosph ate with 32p is known as -P-32 ATP. INTERACTION OF RADIOACTIVITY WITH MATTER For the sake of simplicity, interaction of radioactivity with matter can be divi ded into two broad categories (0 excitation, and (//) ionization, depending upon the sfrength of the interaction as we will see below. Of the two, ionization constitutes by far the most importa nt interaction. Excitation. Radiation can interact with the orbital electrons of the intervening matter that it is p ..a sing through. This interaction may be weak, capable only of lifting an electron to a higher energy orbital from its ground state. This electron eventually descends to its g round state and the energy difference between the ground state and the higher energy orbital is emit ted as electromagnetic radiation. This type of interaction is known as excitation. lon/zat/on. A closer interaction of radiation with matter can impart so much ene rgy to the orbital electron that it leaves the atom completely. This results in the formati on of an ion pair (a positive atom, i.e., ion and a negative electron). This process is termed/on/zat /oru We can now discuss the interaction of individual types of radiation with matter. (/) Alpha-particles have a considerable energy (3-8 MeV). On account of their gr eat mass, such a kinetic energy means a relatively low velocity (Just 1% of the velocity o f light). These particles therefore spend a relatively long time in the vicinity of orbital elec trons they approach. These particles therefore have a high probabflity of interacting with the interv ening electrons. Moreover, they have a double positive charge and a great mass, All this ensures that they will frequently colllde with atoms in their path. Consequently, alpha particles cause intense ionization/ excitation depending upon how strongly or weakly they interact with matter. Sinc e they dissipate their energy quickly they are not known to be very penetrating. Ordinarily, inte raction of an alpha particle with matter will result predominantly in inizatior (//) Negatrons possess a single negative charge, have a small mass and high velo cities. They interact with matter to cause ionization and excitation in a manner exactly simi lar to that of particles. However, due to their extreme speed and vanishingly small size, their probability of interacting with matter is smaller as compared to a-particles. They are therefor e/ess/onz/n9 but more penetmteu3 tho the Negatrons emitted from a given isotope may have different energies. The reason f or negatrons of a given isotope having different energies was explained by Pauli wh en he stated that the energy of a radioactive event is shared between a negatron and a neutrino. T he proportion of total energy taken by the negatron and neutrino can be different for each disint egration, Neutrinus are uncharged, have a negligible mass, and do not interact with matter . Thus, two types of negatrons, those having a relatively higher energy and those having low er energy (soft -particles) can interact with matter differently, Soj [-par/es may cause more n than (//0 Gamma rays are electromagnetic radiation arge or mass. Due to the above properties and because ility of their interaction with intervening .atoms is rather great distances before their energy is dissipated. This makes with matter in three important ways which lead to production trons can in turn cause ionuion and ex/tatn. (Figure 13.3). and are therefore devoid of any ch of their high velocity, the probab less. These rays therefore travel them highly penetrating. /nteract of secondary electrons. These elec The positron is not long-I/red.02 MeV.ray (1.3 Illustration of the ways Inwhlch ¥-rois interact with matter A PhotoelectrcabsOrpthmwheretheTraytmnsfersallltsenergytoanorbtd electron whlchm oves Photoe/ctr/c absorpt/on consists of the transfer of the ent/re energy of the rad lat/on to a s/ngle electron. This tnteract/on results tn only a part of the energy be/rig transferred to the interact/rig electron caustng its eject/on. ray (Reduced energy longer wavelength) Incident¥ .raY 180.51 MeV 0. Y-raY 0. ff tn excess of 1.02 MeV) 7 e-(negatron} 7 . For pa/r production the 'rays must have energy greater than 1. Compton scatter/n is the most common manner/n wh/ch -radlat/on loses its energy. It comb ines with a free electron and is arm/hflated with -ray em/ss/on.51MeV Figure 13. The ejected electron behaves as a negatron. is transfe rred to both the members of the 'pa/r' as ktnet/c energy. lelat/vely low energy -rad/at/on/ndulges/n such/nteract/ons. The energy of the ray. The electron is then ejected as a photoelectron which behaves a s a negatron.02 MeV. Thee-ray itself gets deflected and travels on with reduced energy and a longe wavelength. The/nclden t ray reacts with electrons and is completely absorbed. Eject/on of a positron and an electron (the 'parr') takes place.488 Bophysica Chemistry e. .(Compton electron) . MEASUREMENT OF RADIOACTIVITY It is because radiation reacts with matter that we have means to detect it. We h ave seen how all mdiatWns cause ionization . Both these pes of interactions wi th matter are .and excitation. The number of ionized particles generated tn the gas by direct interaction (primary interaction) with radiation can be inc reased several by various methods discussed below. necessary to maintain the measuring arrangement constant during ea ch series. There can be various motives behind the measurement of radioactivity. :is. measurement of absolute activity is not necessary as the answers to bioclemical problems almost always be obtained by measuring the activity of the sample relative to th at of a which emits similar type of radiation(s). is compared to the amount r introduced in the body. the amount of radio-element found in a formed by the body or otherwise present in an organ. paramount reason for determining radioactivity is the wish to determine the acti vity of a Here it is important to distinguish between absolute act/v/ty and relative activ ity.isotopes n Bogy 489 The simplest arrangement to measure radioactivity involves measurement of the ' radiation in agas filled chamber. they emit electromagnetic radiation. Method Baed Upon Ga Ionitlon . the electrons are not made to the atom completely. In biochem istry. the relative activity refers to the number of disintegrations accounted for. The scintillation counters. exploit the excitation caused by interaction in order to measure radioactivity. however. activity refers to the number of disintegrations actually taking place in the sa mple. mber of 'counts per minute' (or counts per sec ond). When thes e return to their ground state. Here. For example. The photograplc methods of determation f depend upon ionization action of the rys which makes the grains of sver to the developer. but are merely catapulted to lgher energy levels. The standard usually is a known aliquot of the radioactive ma terial into the experimental system. relative measurement is eas ier the absolute ones. 1. on the other hand. Obviously. This light ca n be/ and the number of such flashes gives an index of the intensity of the radiation. with a certain experlmenal arrangement serves as a measure of the relative activ ity. Fhe nu. beta. Principle of the ion/zatiOn chamber I/es in the measurement of the number Of lon g and produced by the radiation in a as filled chamber.Alpha. This is achieved by reating an potential across the chamber by means of two electrodes. To avoid this recombination a . A.. a > fl > (10. and gamma radiations have different capabilities of ionization... these methods would prove incapable Of detecting¥-ra diation and even B-particles. Ionization Chambers -lonization chambers are not used in biochemistry on a larg scale (moreover. Yet a short he/r mode of action is required for a good understanding of the-proportional and counters.. while the positive long travel tovrds the cathode.000 : I00 : 1) From the above it becomes clear that although it is possible to detect a and par ticles by ionization methods. . which can be measured directly with the help of can be amplified electronically. The electrons then rush the anode. These negative and positive long formed in the gas chamber attract one another and Can leading to formation of neutral atoms and molecules. The order of r to induce ionization decreases in the order. wit h the of seint±llation counters these chambers have now become obsole). The migration of t hese particles gives rise to a current. Applied field Figure 13.4 Relationship between the voltage applied and the number of long coll ected (current]. only to lose it again in a repeat collision. is an unequivocal reflection of the inten sity of radiation and hence a measure of the activity. This physical effect is elaborated below. However.4. At each collision. Proportional Counters As obvious in Figure 13. they lose part or all of their kinetic energy. In practice. The current through the chamber is.d{ferent counters operate are indltxrted. When the current obtained is plotted against the app lied voltage.. the region of saturation current shows up as a long plateau in the value of the current (Figure 13. therefore. a rapid increase in the current is observed. Hence from the original evenJu . B.490 Biophysical Chemistry strong electric field is created which accelerates the particles towards the res pc. The electrons rushing towards the electrodes are involved in several collisions with .4). At each collision. This process is repea ted until the long finally reach the electrode. tive electrodes giving them lesser opportunity to meet and recombine. Now.intervening neutral atoms or molecules during their passage througl the gas. The regions at whlch. they regaln their energy again. then dictated solely by the number of long formed per unit time in the sensitive volume of the gas. ionization by collision (or gas amplification) is responsible for this i ncrease. A new physical effect. the primary ion pair produced du e to radiation ionizes more gas molecules to produce secondary long which are also accelerated and which also collide with other molecules to ionize them. Above a certain minimum va lue of the field strength essentially no recombination takes place. ff the nature of the radioisotope and the e xperimental conditions remain constant. due to the applied field . ionization chamber is always used in the region of this plat eau. the plateau discussed above ends ff the field applied across the chamber is further increased. This 'sammtWn current. the .a whole torrent of long reach the electrodes. This is the principle of gas amplification and is known as the Townsend avalanche effect after its discoverer.4. As is evident form Figure 13. in the proportional coun ter region. As a consequence of this gas amp lification the current flow increases. Fina lly we reach a where the size of pulse is totally independent of the amount of primary ionizati on. This simplicity imparts stab ility to counters. counters operating in this region require a very stable voltage supply since even small anges in voltage result in large changes in amplification. of the original charged particle causing ionization.4) in which the Gelger counter operates. the proportional region Is l eft behind d the sizes of the pulses cease to be proportional to the number of primary elec trons or the 01tage applled. It Is very logical then that the former have replaced the latter. C. Scintillation counter s can also ferentiate between different Isotopes and do not suffer. are therefore nearly equal in magnitude. Th us. The discharge produced by each ray does indeed increase as the v oltage rises. from the disadvantages of the proportional counters. t the relative increase is the smaller.directly proportional to the number of primary e lectrons produced y the radiation until a certain voltage is reached where a plateau occurs. The basic advantage of Geiger counter is derived from the amplification (each pr imary ion yields dose to 100 million secondary ion pairs). The disadvantages of Geiger counters are (/) the necessity of having high voltag e.owever. or -particles give similar pulses. . both the Isotopes can be measured by thes e counters. . Sinc e all pulses are alike. Fundamentals of Geiger Counter When the voltage supply Is still further increased. The amplification is so great t hat an simple external electronic amplifier is sufficient. if a sample s labeled with two different Isotopes. and nability to distinguish pulses due to different physical processes. Proportional counting has the advantage that particles of different energies can be listlnguished by pulse-helght analysis (see later)because the size of the curren t pulse Is roportional to the ener. All produced by any type of radiation. the greater the primary ionization. This is as the Geiger region (Figure 13. which cannot be distinguished from one anothe r.sotopes n Biology 491 mmber of Ion pairs collected is. It takes some amount of time for the ion pairs to reach their respective electro des. During other ionizing particles entering the tube do not produceionlzation and thus esc ape Counter or Anode High voltage Metaltlzed cathode Window Path of a particle . Some of the common quenching agents used are ethyl formate and the halogens. a Geiger-Mfiller tube might gi ve a discharge.492 Biophysical Chemistry detection. however. How does quenching occur? Suppose that the gas filled in the tube is helium. results in a continuous disch arge. the long are neutralized. The process. the detector w ill not to any other particle except the very first which enters the tube. If continuous ionization is not prevented. Usually this is about 100-200 s. Both these radiations are capable of causing ionization and produc e ion pairs. Consequently. the time that an ion pair takes in reaching the electro de is know as the dead time of the Geiger tube. probable that the positive helium ion recombines with an electron and becomes ne utral agair In helium. propane. chlorine. bromine). In order to overcome this the tube is quenched by the addition of sui table gas reduces the energy of the long. The quenching agen t continuous ionization. Butane has a lower ionization potential compared to helium. Thus. Let us try'and understand the importance of quenching. While approaching the c athode. ethanol. From the above it is clear that each quenching event results in the quencher. Suppose butane is present as a quenching agent. The above limitation of conventional tubes can be addressed by what is called a . argon) to which has been added a small amount of a (butane. might "e scape" and produce their own ionization avalanche. This is the s ignificance quenching. This is Most commercial tubes become useless after 10s to 101° pulses. such a recombination process is associated with the production of UV and X-radiations. will give rise to a positive helium ion and an electron. Instead it br eaks the bonds of butane resulting in the destruction of the molecule itself. occurring again and again. Now the positive hel ium with a butane and physical and chemical properties of butane are such that exces s recombination is not thrown out in the form of UV or X-radiations. The tube is usually fille d with inert gas (helium. Some of these tons. Upon reaching the electrodes. The Geiger tube therefore should become useless after some time. if care is not taken. neon. ethyl formate. To this process we need a quenching agent. other !-particles may also lose sufficient energy in penetrating the occur if the window is removed and the measurement will have great This modification is known as the windowlessJlow counter. The thickness of the window can vary according to the isotope being measured (th in window mica or mylar for soft [-emitters such as4C. The end window tube is the most widely used type for solid sample s.5(B). (i) Solid mll. the window here is eliminated for greater sensitivity. L/qu/dflow type of tubes. A few popular types of Geiger-Mfiller tubes are illustrated in Figu re 13. (i/ L/quid tp/ Use is generally made of the Annular well. Geiger are still in use. Counter tub. . Thin wall dpplng. they can be used to measure high energy -emitters only. Although they are gradually being phased out by scintillation counters. This is done to allow those -particles which have very low energies and cannot enter even a thin windo w.Such a tube has entrance and exit ports through which gas is madd to flow contin uously. whether it is soft or high energy -emitt er etc). Since these are all made of glass. A few more details about the counter tubes and counter charact eristics therefore provided below. The choice of counter tubes to be used depends on the nature of the sample to be (whether the sample is solid or liquid. thick window of glass for high ener gy such as P}. Mica etc. metal cathode or metal window for liquid sample End window (Glass.493 Insulated base Sample bead Insulated internal .) (i) (fl) (fli) base cathode I Insulated base Long glass tube Anode '.cMaetolldie efu rface . metal cathode Insulated base Internal. needle probe type is in current use to locate im plants or of radioisotopes in several types of diagnostic tests. (v) Thin-tvall dipping type.(iv) (v) (vl) 13. (vO Needle probe type.. The precise the plateau differs from tube to tube but is generally about 300 volts. the starting potential. The characteristic curve at first sho ws a hike in counting rate. In this region only high energy B -particles tube discharge. not caused by radioactivity.is the most popular. If the p otential is further raised. Although. The tube should never be allowed to operate in this continuous disc harge since it can lead to serious tube damage.6 illustrates a plot of count rate from a given radioactive source against potential applied to a Geiger-Mtller tube. counters are preferred for such isotopes. (iii) Annularwell type. Here the potent ial electrodes is so high that spontaneous discharges. The tube attached to a suitable can be moved along the laboratory table or other surfaces to locate the region o f (/v) D/agnost/c. (iv) Liquld-flow type. (ii) Thi n-wall type. in the tube. This hike begins to taper and finally levels off into a plateau. The very thin.5 (B) Different s types of Geger-Mfiller tribes: (0 End-window type. another sharp rise in the count rate occurs. . can be used to measure soft B-e mitters. Characteristics 13. Thin wall tubular type. gasJlow counters which are windowless. (iii) Spillage. For en ergy emitters such as P. However. nearby X-ray sources . background radiation might be reduced by shielding the instrument with lead. care sho uld to ensure that all the samples have the same surface area. Steel planchets or ground glass discs are generally used fo r solid samples before they are counted with a thin end window tube. Secondly. value of background count must always be subtracted from the actual count. ff the position of soft t-emitting such as "C and varies by just one mm the count can vary by as much as 5%. most commonly the source of this background count is the circuit noise. (ii) Oomtrle q0"eCt. Thus. (No j ¢flmcwltom Consider a sample which is quite thick. particles emitted below a certa in distance are absorbed by the sample itself and never reach the counter.6 The plateau of Gelger-Mller tube: the tube s operated in this regton. This is so soft -emitters like C. Geiger Tubes: Certain Practic Aspects Following points should be remembered while counting radioactivit (0 Background count. A Geiger counter operated even in the absence of a sample will register some counts. it is best to measure soft 3-emitter by scintillation counting. This is known as serf absorptlon and the th/ck. The radioactivity from surfaee will have to traverse the intervening air between the sample and the win dow. In such cases. This is known as the background count. or However. This problem may be partially overcome by making samples as thin as thin). oth erwise fraction of the emitted radiation entering the tube may vary and may cause large errors. automatically subtract the background count from the experimental value. This co uld be du many factors such as natural radioactivity in the vicinity. then the air and window before it enters the tube. Such samples always be placed in more or less the same position with respect to the tube. . geometric effects are higher for soft [3-emitters. the radioactivity from its center will have to traverse the solid above it. How ever. geometric effect is not significant.Region of Threshold potenti continuous discharge 0 800 I000 1200 1400 1600 1800 Volts lgure 13. Photorphi© Method To photography we owe the discovery of radioactivity. The photographic emulsions nowadays for detecting radioactivity contain 80% or more of silver bromide so th at lltfle radiation energy is lost by absorption in the gelatin and the grains of silver a re in dose .2. in Biology 495 Ionizing radiations act upon a photographic emulsion to produce a latent image m uch as The radiation interacts with the emulsion to produce electrons which reduce halides to metallic silver. chromatographed. If the further objective is to deter mine which type of cell(s) within the organ is involved in accumulation of the isotope. A radioactive isotope of activity has been administered to an organism. The blackened area on tire film wou ld which has taken up most of the radioactivity (from our knowledge !anatomy we can easily identify the organ). merely catapults it to a higher energy orbital of the atom concerne d. from when the electron descends to the ground state. when excited. The cell sin the are not involved in the 'trapping' the isotope would show up as light spots. rather t han an electron. known as fluors. The different components will out on the chromatogram after a suitable time. 3. the cells are separated form the organ. it emits an electromagnetic radi ation. and after some development. whe reas e cells which take up the isotope would show up as dark spots. If we further wan t to determine cell chiefly takes up the isotope. the organ question is excised from the animal and wrapped in a photographic film. black spots will appear onthe film exactly at the position of component in which the isotopic label appears. fluoresce. Consider the following example. If this chromatogram is now broug ht icontact with a photographic film. This phenomenon of fluorescence . A photographic film is wrapped arou nd the After a suitable time the film is developed. This metallic silver during development shows up as black It is possible to measure the absorbed dose of radiation by densitometry of the blackened Although photographic method is no more used for counting radioactivity in routi ne samples task is increasingly being performed by scintillation counting). it still has a speciallzed known as autoradiography. Methods Based Upon Excitation We have already discussed excitation to be a process where a radiation.Our first objective is to ascertai n tissue 'traps' more of administered isotope. of Scintillation Counting Scintillation can be counted by two different techniques (Figure 13. Obviously. Or/the other hand. the densely packed atoms in a crystal provide of collision. This since the y-rays are electromagnetic radiation and only rarely collide with neig hbouring or excitation. the radioactive sample is suspende d in a system composed of the solvent and an appropriate scintillator. The light emitted in scintillation c an be 14) which converts the photon energy into electrical pulse whose magnitude remains proportional to the energy of the origl nal radioactive fact that the pulse magnitude is proportional to the energy of the original radi oactive is a considerable advantage of scintillation counters over Geiger counters. which in turn is placed adjac ent photomultiplier is connected to a high voltage supply and a scalar.7). sc.emitters (3H. anthracene for g-emitters).intillation counting is particularly usef l for measurement of y-emitting iso topes.the sample is placed close to a fluor crystal (crystallized zinc sulphi de for sodium for y-emitters. solid scintillation counting is not so satisfac tory :weak g-. C. . S) since even the highest energy negatrons have a very low The soft gemitters will thus fail to cause sizeable ionization or excitation in In liquid or internal scintillation counting. Liquid counting is extremely useful for quantitating soft g-emitters. enab ling counters to detect and measure two different isotopes in the same sample. In solid or externa/ counting.by radioactivity is known as scintillation. This transfer of energy is most efficient if an is used. A. the radioactive sample is dissolved or suspended in a scintillati on composed of solvent and primary and secondary scintillators.7 Solid and liquid scintillation counting.8} imparting a disc rete its energy to the solvent molecule.1. however. a schematic illustr ation. 4C. Since most of the radioisotopes used in-biology are soft [-emitters (3H.8 Light Ught cover Sample Lead shieldhng Sample + fluor Biophysical Chemistry Light tight cover Photomultiplier High voltage Scalar High voltage Scalar supply 'supply Solid scintillation Liquid scintillation Figure 13. It may. The radiation from sample molecules collides with a solvent molecule (Figure 13. we discuss liquid scintillation counting in reasonable details. Collision with solvent molecule and concomitant loss of energy which perpetuate until the radiation has lost most of its energy and is captured . 35S ). be pointed out much of what transpires for liquid scintillation counting applies equally well t o solid counting. Liquid Scintillation Counting As said above. Different solvents with differing relative efficiencies of energy trans fer are listed Table 13.496 Figure 13. It @ e-----O----. (R) represents radioactive compound. e" represents emitted H-particle..e------O-. @ indicates solvent molecule in grou nd state. and represents excited solvent molecule .e 200-aO0 nm Primary fluor Secondary fluor -d 330-400 nm 400-480 nm Energy transfer in liquid scintillation counting and subsequent fluor excitation . Int.3 dlmethoxybenzene 80 Xylene 97 Methox-ybenzene (anisole) I00 Toluene 100 Data adapted from J. 2 :1 (1957). The is due to the peculiar chemical nature of the solvent.1 Relative Counting Efficiency of Some Representative Solvents for Scin tillation Solvent Relative counting efficiency% Ethanol 0 Acetone 12 Ethyl glycol d/methyl ether 60 1. This process is known as phosphorescence . D. The chemical configuratio n with aromatic ring structure is such that they have particular electrons. Table 13. Appl. The number of collisions with the solvent molecules is a function of the Initial radiation energy. Hence.Isotopes n liology 497 -noted that only about 5% of the total radiation energy. Felgelson. the amount.4 d/oxane 70 1. The rest of the energy is released as heat. The solvent emits light of a very short wavelen gth which . The solvent molecule. which has become excited as a result of collision with the radiation. Davldson and P. Radial Isot. light as it comes back to ground state.. the which are relatively loosely bound to carbon atoms and can be raised easily to e xcited by radiation (also see Box 8. J.of light emitted is directly pro portional to the radiation energy.4).is finally observed as l ight. The structures of popularly used primary fluor ) and secondary fluors such as bis-MSB and dimethyl-POPOP are given in Figure 13 .9. a second molecute is added to the system. absorbs light in the range of 260nm corresponds to the emission spectrum of the solvent} and emits light of a longer (340-430 nm). Just like the solven t. The secondary fluor absorbs light emitted by t he fluor and emits light with a maximum in the visible region. This longer wavelength light can be measured by most of th e modem However.9 Structures of popular prlmo and secondary flwars . This range is too short to be detected by most exist ing To circumvent this problem.in the range of 260-340 nm. Such a shift to a longer wavelength can be easil y obtained what is calied a secondary fluor. older generation instruments are not sensitive to even this wav elength need an even longer wavelength. the nature of the primary and the secondary fluors is dependent on their aromatic and the availability of the -electrons. This known as a prtmaryfluor (also called sc/ntt//ators). CH:CH-H: CH CH3 bls-MSB CH3 CH3 dlrnethyl-POPOP CH3 Figure 13. (v) Sample preparation for scintillation counting is generally devoid of com plexities and in any case easier than required for Geiger counters. suspensions. Advantages of Scintillation Counting Advantages of scintillation counting over Geiger counting are discussed below. (iii) Geiger counters cannot differentiate between radiations of different iso topes and so cannot be used to measure dual or triple labeled samples. 50% efficiency is quite routine with scintillation counters. Frequently different kinds of accessories may also be required. gels. virtually any kind of sampl e (liquids.498 Biophysical Chemistry Very small amount of light is yielded by the fluors. solids. Most of the commercially available inst ruments use atleast two photomultpliers (for a detailed discussion of photo multipliers. while only 5% efficiency is possible in detecting 3H by G eiger counters. Consequently. (iv) Since fluorescence decay is very rapid compared to the dead time in Geig er-Muller counters. (/) It has already been discussed that soft B-emitters are not satisfactoril y detected by Geiger counters. see Chapter 8). a highly sens itive method of detection is necessary to measure it. However. On the other hand. These kinds of samples can be measured easily in a scintillation counter. Disadvantages of Scintillation Counting Most of the disadvantages in scintillation counting have been overcome by modifi cations in the instrument design. Some of the important disadvantages are liste . emulsions. (//) It has been seen in the preceding sections that different types of Geige r counters are required for measuring different types of samples. etc. The photomultiplier converts the light energy into an identical electrical signal th at can be easily manipulated and measured. much higher count rates are possible in scintillation counter. Thus.) can be accommodated in a scintillation counter and its radioactivity can be determined accurately. chromatograms. (it0 The high voltage applied to the photomultiplier gives rise to electronic events in the system which are not dependent on radioactivity but which contribute to a high background count. Alternatively. this results in product ion of a pulse of reduced voltage. the number of photons -reaching the photomultiplier per B-particle is reduced. In either of t he cases. it is being separately discussed slightly later. A sample can at times contain substances which may absorb th e B-particles without emitting any photons. This o ccurs when the energy transfer process. (/) The greatest disadvantage of scintillation counting is quenching. Chemical Quenching. is interfered with at any point. t he sample may contain substances that do not eat up the B-particles but absorb the photons emi tted by the fluors as a result of the latter's collision with the -particles. They are (/) chem ical. (ii) color. Quenching As pointed out before. In other words. Because of the importance of quenching. The three are discussed below briefly. and (///) dilution quenching. There are basically three types of quenehtng. quenching means a reduction in the e/lclency of transferr ing energy from the -particles to the photomultiplier. (ii) The cost per sample of scintillation counting is significantly higher than Geiger counting. described previously.d below. This effect is known as photomultiplier no/se and can be reduc ed by cooling the photmultiplier. This means that these particles do not excite the fluor and get 'eaten' up even before they can reach the fluors. sufficient . chlorinated hydrocarbons. Thus the enduring d efinition of color quenching remains absorption of photons from the secondary fluor. aci ds. In addition to the above there is another kind of quenching which has been calle d opt/ca/ quench/ng. Determination of Counting Efficiency Counting efficiency may best be described by the mathematical relationship Counts per minute of the radioactive standard . This is just what the title says. This is caused by the dflu tlon of the fluor by the sample.× I00 Isotopes in Biology 499 energy does not reach the detector and a weak pulse may be obtained which is not a true picture of tile energy of the radioactive particle. Chemical quenching can often be tackled by increasing the concentration of fluor s in the scintillation cocktail. dissolved oxygen. Dirty scintillation v ials may interfere with light emitted by the sample and prevent its passage to the detector. This should not be taken to mean that if there is no color visible in the scintillation cocktail. This results when certain substances in the sample can absorb s ome of the photons emitted by the secondary fluor. It can be corrected by correcting the data obtained for the dilution fa ctor. Co/or Quenching. i t is free from the menace of color quenching. and peroxides. High dilution will reduce the probability of scintillation event Naturally. D/lution Quenching. This que nching can also be reduced by increasing the fluor concentration. salts. this type of quenching normally takes place when working with liquid samples. or brown normally cause severe quenching. red. Such a process is called che mical quenching. The secondary fluor emits in the UV regions also and color quenching can result through absorption in that region also. It is called color quenching main ly because substances that are yellow. In this the sample components do not interfere. Commonly encountered :chemical quenchers are water. Counting efficiency dep ends upon several factors. A radioactive standard of a ccurately known activity is essential for the determination of the efficiency counting. The counts that we get n ow may be .this sample a fixed amount of interna l standard of known disintegrations per minute is added. Suppose that the sample gives X counts]minute. in practice. The. use of radioactive standards is in principle similar to that of standards in colorimetric or spectr ophotometric assays. provid es a reference standard). However. Samples contain ing same concentration of radioactive samples might produce different number of counts an d this discrepancy is expressed in terms of counting ejiciency. st ability. the one major which has to be taken care of. it is necessary to determi ne the counting effieiency before one can compare different samples. the efficiency frequently varies. when dissolved in unquenched scintillation mixture. The sample is now counted again. All these factors can be minimized with due care during the experimental set up. temperature of the counting chamber and the volume of fluid in the counting vial . Suppose again that the disintegration s of the internal standard are Y/minute.Disintegrations per minute of the radioactive standard It will be a fallacy to believe that a given amount of radioactive sample will g ive the same count rate whatever be the conditions under which it is counted. some of which are dependent upon the instrument sensitivity. and (tl) external standard (a y-emitter incorporated in m ost instruments). (1) Internal standardization. To . In this method the first gtep is to count the samp le. is the quenching (see above) which varies with di fferent Since. There are two types of standards (0 fntema/standard (this is usually a 1 3-emitter of known activity which. Its count rate is first deter mined in each of a series of quenched standards of known counting efficiency. (v) It should preferably be in liquid form so that small amounts can be dispense d easily. (ii) ¢terna! standardizati¢n. The y-ray from the external standard pene trates the counting vial and gives rise to scintillation. With the advent of the scintillation counters with fully automated standard faci . The external standard is always a y-ray emitter built into the liquid scintillation counter. The count rate of the external standard is then related to this efficiency. The drawback of this method is that it is time consuming (one has to count each sample twice) an d requires accurate pipetting of the standard (failure in this might lead to a large error) . The activity of the external standard is irrelevant to the preparation of a cali bration curve as it is used merely as a source of radioactivity. Its usual position in the instrument is far aw ay from the counting chamber. The external standard count rate ob tained in a sample of unknown activity can then be used to determine the efficiency of count ing for the particular sample. (No It should be of a high specific activity. it is aut omatically brought adjacent to the counting vial. (/) It should be readily soluble in the scintillation mixture. Internal standardization is an extremely simple and at the same time reliable me thod of determining counting efficiency and thus to correct for all types of quenching.X).500 Biophysical Chemistry denoted as Z counts/minute. (i/) The amount of standard used should not cause quenching.x 100 Y The internal standard used should have the following desirable qualities. (iv) It should be chemically stable. The counting efficiency of the sample can now be cal culated by the relationship Counting efficiency = (Z . when required for efficiency determination. However. howev er. This graph known as the channels rati o quench correction curve is utilized to determine the counting efflciencies of experimen tal samples. be pointed out that the internal standardization is a more accurate method. The counting efficiency in chann el A would then be [{Y/X) x 100]. (ii] Channels ratia. Further addition of quencher is made and the sample is recounted in both the channels. It might. A sample containing a known amount of standard (X dpm) is prepared and counted in both the channels. The ratio of the two channels will be Z/Y. it is not mandatory since e ach sample can be counted twice at different channel settings in a single channel apparatus. Quenching reduces the average energy level of the I-spectru m. Aga in the counting efficiency and the channels ratio is determined. Moreover. This re lationship is utilized in the channels ratio method of determining counting efficiency. One o f these channels is set to cover the entire unquenched I-energy spectrum. After many such additions of quenching agent and calculations of counting efficiencies and channels ratio at each stage. . while the second channe l coves only half or a third of this spectrum.lities.. this technique has become advantageous over the internal standard method in as much a s that the counting efficiency for each sample can be determined automatically. This decrease in I-spectrum energy increases with increase in quenching. Althou gh a two channel scintillation counter is preferable for this method. Some known am ount of quenching agent is added to the sample and it is recounted again in both the channels. counting efficiency values are plotted against channels ratio values. t he errors due to pipetting are not there and the method does not consume tlme. Let us assume that channel A gives Y counts per minute and channel B gives Z counts per minute. Isotopes in BIogy 501 Thus. It is also less time consuming since only one count is required in two-channel scintillation counter. The curve. channels ratio for each sample is determined and the efficiency can then b e read off the graph. Moreover. The channels ratio method is satisfactory for all types of quenching. it is the most accurate method for cotmting efficiency determination. however. For different isotopes and scintillators different calibrat ion curves have to be prepared. applies only if the same isotope and scintillator is used m the experimental sample. . Moreover. (0 It should be readily soluble inthe scintillation mixture. The y-ray from the external standard pene trates the counting vial and gives rise to scintillation. this technique has become advantageous over the internal standard method in as much a s that the counting efficiency for each sample can be determined automatically. (i/0 It should be of a high specific activity. when required for efficiency determination. (v) It should preferably be in liquid form so that small amounts can be dispense d easily. The drawback of this method is that it is time consuming (one has to count each sample twice) an d reires accurate pipetting of the standard {failure in this might lead to a large error) . Internal standardization is an extremely simple and at the same time reliable me thod of determining counting efficiency and thus to correct for all types of quenching.500 Biophysical Chemistry denoted as Z counts/minute. However. The count rate of the external standard is then related to this efficiency The external standard count rate obt ained in a sample of unknown activity can then be used to determine the efficiency of count ing for the particular sample. The ctivity of the external standard is irrelevant to the preparation of a calib ration curve as it is used merely as a source of radioactivity. (iO xternal standardizagiom The external standard is always a y-ray emitter buil t into" the liquid scintillation counter: Its usual position in the instrument is far aw ay from the counting chamber. it is aut omatically brought adjacent to the counting vial. (i0 The amoun of standard used should not cause quenching. t he errors due . With the advent of the sclnt111ation counters with fully automated standard faci lities. The counting efficiency of the sample can now be cal culated by the relationship Counting efficiency = x 100 Y The internal standard used should have the following desirable qualities. Its count rate is first deter mined in each of a series of quenched standards of known counting efficiency. (iv) It should be chemically stable. This decrease in Is-spectrum energy increases with increase in quenching. it is not mandatory since e ach sample can be counted twice at different channel settings in a single channel apparatus. It might. are plotted against channels ratio values. . Let us assume that channel A gives Y counts per minute and c1anne] B glves Z counts per minute. This graph known as the channels rati o quench correction curve is utilized to determine the counting efficiencles of experimen tal samples. Quenching reduces the average energy level of the Is-spectr tlm. The counting etclency ]n channel A would then be flY/X} x 1001. The ratio of the two channels will be 7-. Aga in the counting. Further addition of quencher Is made and the sample Is recounted In both the channels. This r elationship is utilized in the channels ratio method ofdetermlning counting elciency. (iii] Chtmael ratio. howev er. After many such additions of quenching agent and. One o f these channels is set to cover the entire unquenched IS-energy spectrum. while the second chann el coveks only half or a third of this spectrum. calculations of counting efficiencies and channels ratio at each stage. Although a two channel sctntt//ation counter is preferable for this method. be pointed out that the internal standardization is a more accurate method. efficiency and the channels ratio Is determined.to pipetting are not there and the method does not consume time. counting efficiency values. A sample containing a known amount of standard (X dpm) is prepared and counted in both the channels.. Some known amo unt of quenclrg ' agent Is added to the sample and it is recounted again in both the channels. Moreover. For different isotopes and scintillators different calibrat ion curves have to be prepared. charlllels ratio for each sample Is determlecl ad the ettclency" can then be read olT the graph. It is also less time consuming since only one count is required in two-channel scintillation counter.Isotopes in BWlogy 501 TIUs. applles only Lf the same isotope and sctnt-fllator is used In the experimental sample. . it is the most accurate method for counting efficiency determination. The curve. The channels ratio method is Satisfactory for all types of quenching. however. The nature of the sample to be counted dictates the mode of sample preparation f or liquid scintillation counting. when mixed with water form gels suitable for scintillation counting.5O2 Biophysical Chernist3 Sample Iepaation for BeimtIIIatlon Comt For solid scintillation counting the sample only need. (//) It should have least possible quenching ability. Contamination of solvents with chemiluminiscent substances might produce large e rrors in counting It is therefore necessary that the highest purity solvents and solut es should be used in all iquid scinti]lation counting.4.4-dioxane are in use. and uniform. Generally. toluene based cocktails are preferred. suspensions or gels may be satisfactory. be transferred to a plast ic or glass counting vial. There is a very large range of scintillation cocktails ( mixtures) available. For aqueous samples. for non-aqueous samples. A good cocktail should have the following desirable properties. each useful for a particular type of sample. (iv) Its components should be reasonably stable. in certain cases. Cocktail for toluene soluble materials: . colourless . Samples may be prepared in such gels also. addition of a second solvent to toluene may make toluene-based co cktails suitable for aqueous samples. Some examples of toluene and 1. ('/) It should be inexpensive. Alternatively. Compounds such as Triton X-100. Keeping the samples in dark before cou nting is advisable in cases where chemiluminescence might pose a problem. However. cocktails based on 1. This is even more neces sary when one is measuring soft -emitters. (/) Even after addition of radioactive sample it should be clear. Its quality should not deter iorate on storage.dioxane based cocktails are provided below. . Soluene. This. solid samples. Radioactive material adherent to solid supports such as glass fibres.2g POPOP + 1. (//) Toluene based cocktail suitable for aqueous samples: 500 ml toluene + 500 ml Cellosolve (2-ethoxy-ethanol) + 5g PPO or 7g butyl-PBD. If the compounds are separated by gel electrophoresis they may have to be eluted or extracted from the gel by slicing. lg POPOP or dimethyl-POPOP/litre. grinding and centrifuging after addition of s uitable buffer.4-dioxane to make the volume 1 litre. Alternatively. the label can be collected in the form of 4CO2 which can be subsequently co unted. discs. chemiluminescence may pose a problem. lg POPOP or dimethyl-POPOP/litre. or Protosol). If necessary. t ml of water mixes quite homogeneously with 10 rnl of cocktail. like plant and animal tissues are frst solubilized in strongly basic solutions (Hyamine 10-X hydroxide. add 0. the sample has to be bleached before count ing. NCS solubilizer. If the label is 3H. add 0. it can be converted to 3H20 which can be counted. A good alternative to bleaching is the combustion of sample. chromatography paper or filter paper may be placed as a whole in a glass vial. (i//) Toluene based cocktail suitable for aqueous samples: 667mi toluene + 333 ml Triton K-100 + 5g PPO or 7g butyl-PBD. Ig POPOP/lltre toluene. A simple cocktail is then added to it and the mixture counted. may not give a good reproducibility. scintillation cocktail is added and the sample can be co unted. If color quenching becomes a problem. 3 ml of water mixes quite homogeneously with 10 ml of cocktail. Once the solubilization is over.503 Isotopes in Biology If necessary. The supernatant can then be mixed with a scintillation cocktail. If thelsample is la beled with 14C. however. (iv) Dioxane based cocktail for aqueous samples: 100 ml of absolute methanol + 20 ml ethylene glycol + 60g naphthalene + 4g PPO + 0. If necessary. 1 ml of water mixes quite homogeneously with I0 ml of cocktail. Here also. add 0. . Care has to be taken that the bleaching process does not give rise to chnflumine scence. For dual isotope analysis. It is evident from the figure that the energy spectra o f two isotopes (A and B) are almost non-overlapping. with the channel set differently each time. it is desirable to have a two channel apparatus. and measure one isotope at a time. Figure 13. It is. Alternatively one can make do with a single channel scin tillation counter. . 3P and 14C. The two ch. aH and asp.Double Isotope Analysis As mentioned above. S and asp.annels (a puls e height analyzer consisting of a threshold and window is known as a channel) will now measure the isotopes A and B simultaneously. The trick is to set one pulse height analyzer to reject all p ulses of energy below W (threshold W) and above X (window X) while the second pulse analyzer is set to reject pulses of energy above and below Y and Z respectively. aH and S. scintillation counters make it possible to measure two isoto pes in a single sample. There are many such pairs of isotopes who se energy spectra are very different.10 illustrates the prin ciple of double isotope analysis. necessary that the energy spectra of the two isot opes being measured ar sizeably different from each other. however. and even two isotopes of the same element I and I. For example. Isotope A ' I Isotope B I Pulse energy Figure 13. Although measurement of stable isotopes is more cumbersome than that of the radloactlve isotopes. USE OF STABLE ISOTOPES IN BIOLOGY Since radloactlvlty Is a magnltude so easlly measurable. However. If subjected to a magnetlc field. These Long. Thls Is achleved by bombardt ng the sample with electrons. there exist n o radloactlve isotopes! with a long enough life tlme (half-llfe) of such Important elements as nltrogen and oxygen. All the long produced have a posltlve charge. Thus.10 Diagrammatic ustmtlon of the p of double . studles Involvlng Oxygen and nltrogen have to use the stable isoto pes of these elements rather than the radloactlve ones. Thls Is the basls of sepa raUon by mass .botope anolyss. The property of Isotopes havlng different atomic masses by vlrtue of their havlng different n umber of neutrons Is ut111zed In the measurement of stable isotopes. It can nevertheless be performed with a very high efflclen cy. Sltrometay Mass spectrometry involves Ionlzatlon of the parent molecule to glve poamt on an d fragmentatlon of these Long to glve fragment ons. use of radloactlve isot opes has galned an upper hand over the stable Isotopes tn blology. wfllbe defl ected to dlfferent degrees depending upon their mass/charge (m/e) values. whlch possess a certaln amount of klneUc energy. The technlque of choice is ma ss spectromet. will depend only on the mass of the ion which is the only basis ng. The number of long of a g iven mass impinging on the detector is a measure of the abundance of that particular ion. only one e lectron is knocked out per atom (there are exception to this rule.1 I ). The mass spectrometer will then be forced to separate the different e basis of their masses. The trick in determining the relative abundance of an isotope label is to prepar e the long in such a manner that all the long have a singl positive charge. field or the accelerating voltage the ion trajectories may be varied so that fragment long of a given rtY e value impinge on the collector slit of the detector. By varying either th e magnetic. They are ac celerated to a constant velocity in a vacuum by a series of negatively charged electrostatic plates and are then deflected from their original trajectory by a magnetic field (Figure 13. which is the basis of separation of ometer. the mass]charge value. the long are deflected by agles wh ich are inversely proportional to the square roots of the masses of the long. For long carrying the same charge the amount of deflection is dependent on the mass of the ion.. Electrostatic field trajectory . The charg long in a mass spectr of difference remaini isotopes solely-on th The separation of long according to their rn/e values is as follows. Le. many times an ion loses another electron spontaneously). so that the long of smallest mass undergo the greatest degree of deflection. The long produced then will differ only in e being equal. lf the charge is kept constant.Ionization chamber Amplifier reservoir Recorder fsotopes tn Bk>logy 505 spectrometry. their mass. the of fall of a drop of water containing deuterium (heavy water) of standard volume through given height of a suitable immiscible liquid of slightly lower density would be slightly more that of normal water.Light long : field long Towards vacuum pump Fure 13.10764 compared to water at 25°C. instead of hydrogen.11 Basic components of a mass spectrometer Falling Drop Method for Deuterium Measurement Deuterium is the stable heavier isotope of hydrogen and the density of "water" deuterium. is 1. Thus. This phenomenon is utfllzed to measure the deuterium conte nt of a . With both these liquids. There are very few liquids immiscible with water that have both the proper densi ty and viscosity to make them suitable for this purpose. that while such labeled compounds remain chemically identical to slmilar compoun ds within the system with which they will mingle. the pathway. However. If such compounds are metabolized. above 7%. samples with higher deuterium cont ents can be suitably diluted. (Refer chapter on viscosity for mathematical treatment of the phenomenon and instrumentation). This is the basis of tracer studies and/the experiments involving this principle . The liqu ids that have been satisfactorily used are ortho-fluorotoluene and meta-fluorotoluene.sedimented throu gh a density gradient (usually CsCI) they will sediment faster than the sample containing the lighter isotope. they differ from unlabeled compounds in as much as that they emit easily measurable radiation or have slightly different masses.506 Biophysical Chemistry given biological sample. they will hand over the radioactive or stable isotopi c label to the next metabolite in the series. Density Gradient Centrifugation If biological samples containing the heavier stable isotope are. THE TRACER TECHNIQUE What is tracer technique? The reason behind biochemists' penchant for introducing radioisotopes in a syste m is. Thus solutions of CsCI have been used to distinguish between DNA with and withou t 15N in its molecular structure. the former i s used at 26.e.8°C and the latter at 19. A single pure compound is nece ssary so that there will be no change in the density due to differential evaporation. the water samples from biological systems usually have low deuteriarn contents. i. the relatlon between the rate o f fall and density appears to be linear upto about 3% D20. and provide the biochemist with a unique possibility of tracing their history and thaLof their successors through a bioch emical pathway. the departure from lin earity becomes significant and the method fails with these liquids.8°C. A biochemist can thus keep tabs on such labeled compounds and trace them in a system at a given t ime. .are known as tracer experiments. 5O8 Biophysical Chemistry General Tracer Requirements The tracer studies in general consist of : () Preparation of a labeled compound (//) Introduction of the labeled compound into a biological system. . and (i//} Separation and determination of labeled species in various biochemical f ractions at a later time. if a compound is la beled such positions. Some of these criteria are listed (i) The starting concentration of tracer must be sufficiert enough to withstand dilution in the of metabolism. Similarly . however. This dilution poses two problems. Suppose sample of labeled CO2which contains 5mg as Carbon. The organism chosen is yeast. Thus. on e molecule will take part in a metabolic reaction. the label must retain its ppsition in the portion of the in which it was originally incorporated. its in the metabolic reaction would be so little as to be almost negligible. through fermentation of glucose. The 4C label has becom e 400 times. What thiSactually means is that for every 400 unlabeled molecules. Let us review the above situation. it might lose its label before it undergoes a metabolic reaction (Figure The tracer will therefore yield no useful information about the metabolic pathwa y. as the time elapses the radi oactive progressively dilute and if the initial concentration was not sufficient enough stage might be reached where assay of radioactivity becomes a problem (radioacti ve are very sensitive and this problem is therefore not so important). The second is of much importance. Of . exchanges between two positions of the carboxyl group. Firstly.in Biology There are. 2000 mg of car bon the form of carbon dioxide evolves. If the level gets further dilut ed. The 'active' hydrogen of alcohols. This is since no information about the metabolic reaction will then be afforded by the (ii) Throughout the metabolic pathway. to an actively fermenting yea st During the course of experiment. 509 certain criteria which the label should satisfy before results be correctly interpreted. amin es and rapidly exchanges with hydrogen long in the surrounding aqueous media. Suppose that we are using a I+C labeled carbon dioxide to determi ne the of carbon dioxide during fermentation of glucose. The original sample of 14C02 (5 mg) has now become ) times. Thus. while C is usefu l for shortexperiments.if one wants to study exchange reactions. the tracer isotope of a givenlelement varies. "C loses its by a factor of about I000 in as short a time as 3. one might not get any useful tracer has been removed through decay.5 hours. liii) The half-life of the tracer isotope sho/hld be sufficiently long. loses its labe l quickly. it is these positions one should isoto pically Figure 13. In long term experime nts. if one has introduced a compound labeled with "C. it might not be very Useful for longterm experiments. As an example let us consider the following. Dependin g on the length of II experiment. On the contrary. The specific acti vity of . it will have suffered no diminution in its activit y even after a long Such labeled compound will then afford much information about the desired metabo lic The sensitivity of tracer methods is their most significant advantage over all o ther chemical physical methods.12A compound labeled in an atom which can be exchanged. ff one has used label (half-life 5000 years). (one has to consider the dilution the activi ty would in the tissue).3. The activity should be so chosen that it is the minimum necessary to permit reas onable rates in the sample to be analysed. as is obvious. and almost non-exlstent for alp and ap. is three times heavier than IH). the effect would be smaller in the case of 2C and 14C. certain restrictions abo ut th. Thus. Whatever we is true for stable isotopes also.7. distribution coefficient of deuterium between water and gaseous hyd rogen 25°C has a large value of 3. If an unnecessarily large activity is administered. understand that the isotope effect would be considerably more pronounced in the case of isotopes of light at oms. Radiotracer techniques can easily detect these substances. therefore. they might do so at different rates. Although of lesser importance than the kinetic isotope effect. Lim/tations of Tracer Experiments There is no known technique without restrictions. One can. the tracer m ay from the experimental organism thereby distorting the results. of (radio) isotopes have also to be appreciated. Another major advantage of using radiotracers (stable isotopic tracers are that the studies can be carried out on a living organism. This implies that a triti um labeled compound can be detected even ff it is diluted 1012 times. for example hydrogen (all.510 Biophysical Chemistry carrier-free tritium is about 50 curies per millimole. Another important restriction is with respect to the activity of the isotope use d. Many metabolic substances are present in tissues at such low concentrations that the most sensitive chemical method is un able to detect them. one should that the equ///br/um/sotope effect also constitutes an important restriction on For example. . These different rates are more or less prop ortional to the differences in mass between these isotopes. whereas that for tritium is merely 6. The heavier isotope therefore more concentrated in water. As compared to th is. being chemically simi lar the are expected to undergo the same reactions. since mass spectrometry is an extremely sensit ive technique and extremely small concentrations of stable isotopes can be assayed with relati ve ease. Although. This effect is termed as the ktnet/c/sotope effect. therefore. Results obtained wi th experimental set up are. We know that the metabolic different concentrations of a given metabolite is different. the normal chemical level of compound in the system is automatically exceeded.When a tracer is added to a biological system.5 shows howresults of radiotracer experiments can be misinterpreted. Box 13. never unquestionable. . uridine is yo ur index of replication or transcription respectively.512 LABELING PROCEDURES Labeling procedures must be given careful consideration if full potential of the tracer technique is to be taken advantage of. That wi ll defeat the whole objective as replication will either not be taking place at all or will be taking place at a rate that won't be much. System Being Labeled Supposing you are using a bacterial culture to study replication or transcriptio n. The label you are using is 3H and incorporation of 3H-thymidine or 3H. Since your aim is to s tudy replication. you must . the culture must not be in a stationary or senescent phase. The consideration to be given can be grou ped under three major categories : (I) the system giving ceils/cell free systems) being la beled. and (3) the labeling format. You must see to it that the culture i s In a state of balanced growth throughout the course of your experiment. (2) the chemical nature of radioactive material. It is better if the culture is in the log phase. Moreov er. and pulse-chase labeling. one should choose the format that is apt. Let us try and understand the method. Pulse labeling is done when the rate of synthesis of a particula r molecule is to be studied. you must ensure that the culture is not making a transition from lag to log phase during your experiment. Labeling Formats By labeling formats we mean the ways of labeling cellular constituents. you must choose the system in a phase that fits your requirements and that the system continues to remain in that phase for at least the period of your experim ent. and a third cell may convert it to uracil and then take up uracil. It may be a better idea to provide labeled uraci l in the third case. These formats are known as pulse labeling. Chemical Nature of the Radioactive Material Different cells have different capacities to take up the extracellularly provide d label. There ar e basically three different labeling formats and each yields a qualitatively diffe rent information. the lag time will correspond to the time that has been spent by the cell to convert extracellular labeled com pound into a metabolite that it can take up. It cannot therefore be overemphasized that depending on the kind of information needed. Moreover. or what happens to transcription during the transitions. if your objective is to study the effect of senescence on transcripti on. For obvious reasons.Isotopes in Biology 513 ensure that the culture will continue to be in the log phase even beyond the tim e-scale of your experiment. For example a given cell may be completely unable to take up tritium labeled uridine . In the second case the experiment may result in a linear incorporation of the label and in the third case a lag period may be there before incorporation becomes linear. this will ind uce large scale errors in your experimental observations. then you must choose these time s deliberately. In short. In the first case the experiment will fail as no incorporation of uridine in the RNA wi ll take place. equilibrium labeling. . another cell may take it up readily. Pulse labeling. This is to ensure that throughout your experimental course the growt h situation will remain the same. Of course. RNA molecules synthesized before the aliquots of culture w ere transferred to the vessel containing the labeled substrate.e. Let us a lso assume that initially only one RNA polymerase can sit on the operon. let us presume that the synthesized RNA is not being degraded (this of course doesn't happen. Th e time of . After a polymerase reaches the end of the operon. Let us take the system to be an operon. only or part of RNA. do not contain the l abel. but transcription can begin if an inducer is added to the medium. aliquots from th e cnlture are transferred to vessels containing small amounts of very high specific activity s ubstrate and allowed to incubate for a short time. but for the sake of un derstanding theae assumptions are necessary). However. Subsequent to this. Lastly. However.Let the objective of our study be transcription in a prokary0tic system. i. The molecule being studied is then purified from the killed cells and the radioa ctivity in these molecules is measured (in our model the molecule is RNA and the label i s The radioactivity incorporated is then plotted against time. The period of incubation is terminated by killing the ceils. L et us assume that the transcription begins immediately upon addition of the inducer. after 3 s econds of transcription by the first RNA polymerase. it falls offand releases free RNA into the cell. The experimental design is quite simple.addition of inducer is taken to be 0 time. Let us assume that the operon is inducible. The inducer is added to the culture.. will be radioactively labeled where the culture has spent time i n the containing the label. Let us suppose that a maximum of 10 such RNA polymerases can sit on the operon. enough space is available for a secon d molecule of RNA polymerase to sit on the operon and begin transcription. . it is normally not transcribed. Here it must be clearly that the transcription has' started after addition of inducer. Thus the rate of RNA sy nthesis in these 3 seconds is double that of the first 3 seconds. 3 polymerases will be making RNA thus synthesizing 3 units of RNA incorporating 60 cpm. Between each slanting llne the time is constant ( in the case dlscsed in the text. polymerase sitting on the operon) and when we kill the cells and count radioactivity incorporated in the RNA we should get a count of 40 cpm. In the third al iquot.13. two polymeras e molecules are synthesizing RNA. RNA isolated and counted for radioactivity' we would get 20 cpm.Suppose that 1 unit of RNA is made in 3 seconds and tha t one unit of RNA corresponds to 20 cpm. which is transferred to the H-uridine vessel at 6 seconds and allowed to incubate for 3 s econds. Remember that when the first polymerase has spent 3 seconds on the operon. 2 units of RNA are mad e ( 1 unit each by each. Remember that in this ali quot the first polymerase had already made one unit of RNA.13 ). Slanting li nes are the mRNA. Suppose that the first aliquot was transferred to a 3H-uridine vessel at 0 time and allowed to incubate for 3 secon ds. At 0 time only one polymerase can sit on the operon and therefore DNA is being transcribed by just the one enzyme at this stage. Horizontal line represents the operon.30 sec 9 10 514 Biophysical Chemistry Let's now see what happens during the experiment. this RNA is not radioactive (Figure 13. in this aliquot. enough space has been created on t he DNA that a second polymerase can sit on it. Pulse labeling. The rate of RNA synthesis here is triple that of the first aliquot. Solid slanting lines represent radioactive portions of the mRNA while the dashed slanting lines refer to the non-radiooctWe portions. Therefore when the cells are killed. Thus now. 3 seconds). Since incubation here too is 3 seconds. 3 see 6 sec Figure 13. But since the ceils were not transf erred to the radioactive vessel. The second aliquot is transferred to the 3Huridine vessel at 3 seconds and allowed to incubate for a further 3 seconds. . Let's now see what happens during the period between 30 and 33 seconds. 10 polymerase mol ecules are itting on the operon. .This logic Can be repeated 10 times because 10 polymerase molecules can sit on t he operon at a time. The data obtained so far. Thus the totaJ number of polymerase molecules on the op eron Gill continue to be I 0 ( it will continue to be 10 from now on no matter at what tim e we study the system) and the count of RNA synthesized will be 200 cpm only. During the period between 27 and 30 seconds. if plotted Gill give rise to a linea r relationship (Figure 13. The firs t polymerase that sat on the operon at 0 time will now fall off and a new one Gill sit at the beginning of the operon. This means that w eare going to obtain a plateau now and that maximum rate of synthesis has been achieved. 10 units of RNA will be made in the 3 seconds of incubatio n and the count be 200 cpm.14 ). at specific intervals of time. alic!u ots are removed from the system. It should be clear from the foregoing that pulse labeling monitors the rate of s ynthesis of a given molecule. This iS the reason why the lab eling period is kept as short as possible. Note that as the labeling period approaches zero. the observed rate will approach the true instantaneous rate of synthesis {ds/dt).14. we will continue with our previous model 0fthe operon and synthesis of mRNA}. T hus ifa mRNA has a half life of 5 minutes. The system being studied is first incubated with large amount of low/moderate sp ecific activity radioactive material (in this case 3H-uridine. and the radioactivity incorporated assayed. Chart obtained with pulse labeling experiment as described in the t ext. the labeling period will be just 0. Once this has been achieved . The experimental design is substantially different.515 24O Seconds 2OO 120 I 6 12 18 24 30 36 42 lgure 13. the molecule purified.5 minutes or less. F. It must be noted that the period of labeling is decided by the half life of the molecule being labeled. Equilibrium labeling measures th accumulation of a molecul e being synthesized. it may be less. . Normally.uilibrium labeling. the inducer is added to start the experiment. the ceils killed. it is no longer than I0 % of the half life. The incubation is continued till the precur sor pool inside the system has reached a constant specific activity. Thereafter. 15 ). will not contain 40 cpm as in pulse experiment.ized between 3 0 and 3 seconds will be the same as that synthesized between 27 and 30 seconds. in the equilibrium exper iment. all the RNA made will be radioactive unlike the pulse expe riment where only that RNA was radioactive which was synthesized during the time the system w as exposed to short pulses of radioactivity (Figure 13. Thus.16 ). but 60 cpm. the amount of RNA synthes. Please no te that tn equilibrium labeling. .Let us see what will be our results with this kind of labeling if we continue wi th our previous model. The second aliquot. This is so because the first polymerase molecule has made 2 units of RNA in 6 seconds while the second polymerase molecule has made I. From t his point on the increment in cpm will be linear (Pigure 13. Supposing each aliquot here too is being taken after intervals o f 3 seconds. After that. The first aliquot will give 20 cpm as only one unit of RNA is made by one polyme rase. however. Total is 3 units and therefore 60 cpm. the initial increments in radioactivity counts at each time interval will be exponent/oL Thi s situation will till the end of 30 seconds. 3 seconds). the preincubation with the label is a critical step in this experimen t. this is never the case. Figure 13. must be corrected for the degradation that is taking place.15. If this doesn't happen. In reality this is just a variation of pulse labeling. t labeling. Therefore. All slanting lines represent radioactive RNA. Two things must be noted here. This is followed by sampling the labeled culture at various times after the . Chart obtained with equilibrium labeling experiment as described i n the text. . Equilibrium labeling. Secondly. It is absolutely necessary that the precursor poo l of the metabolite reaches constant specific activity. we have assumed above that no degradation of the molecule being studied is taking p lace. The data obtained therefore. large-sca le errors will result.516 Seconds Bphysa:al Chemistry 3 sec -6 sec 30 sec ljure 13. Slanti ng lines are the mRNA. In reality. Between each slanting line the time is constant ( in the case discussed In the text.radioactive pulse was i nitially given. Thi s format involves giving the cells a short pulse of high specific activity substrate and then 'chasing' this with non-radioactive substrate (or dflutlng the radioactivity -1000 fold). "ltLe-cha. Horizontal line represents the operon.16. Okazaki interpreted his experimental results in terms of the semidiscontinuous replication model (Figure 13. coli were transferr ed to an unlabelled medium (chase). This perplexin g situation was addressed by Reiji Okazaki in 1968. Both the strands were replicated together. Motion of replication fork 5 . as p er the logic of pulse experiment) had a sedimentation coefficient between 7S and 11S. This would mean that only one strand could be replicat ed at onetime. Also. The inference was clear.3' direction. coli culture for 3 0 seconds with !H-thymidine. This small value can only be given by very small DNA fragments. In his initial experiments Okazaki pulse labeled a growing E. the sedimentation coefficient of the radioactive DNA was much larger.Isotopes in Biology 517 Although just a variation. this format gives a qualitatively different result : it tells us about precursor-product relationship. I1 1 IV)kill IlY'a 5' " Parental strands . Let us understand this with a famous experiment. All known DNA polymerases can only ex tend the DNA in 5' . In another experiment. Subsequent centrifugation of DNA under alkaline condition rev ealed that mucli of the radioactive DNA ( radioactive and therefore newly synthesized. After sometime when the centrifugation was carried ou t. Decades ago it had become known that the two anti-parallel strands of the DNA duplex were simultaneously replicate. the sedi mentation coefficient values increased with the time the cells were grown in the unlabeled medium.17). These small fragments subsequently ca me to be known as Okazaki fragments. how this could be achieved was not understood. the E. The short DNA fragments observed earlier must have beco me covalenfly incorporated into larger DNA molecules. H owever. The evidence obtained with electron micrography and other techniques pointed to the fact that this was not the case. following the 30 second pulse. but discontin uously as Okazaki fragments. the radioactive amino acid would be incorporated primarily in zymogens.3' direction. whereas the lagging strand is synthesized discontinuously. Since most proteins in pancreas are synthesized as zymogens. is also synthesized in 5' .17.3' direction. The newly synthesized D NA strand that extends 5' 3' in the direction of replication fork movement (leading strand ) is essentially continuously synthesized in its 5' . .ljure13. the Okazaki fragments are covalenfly joined together by DNA ligase. The two parent strands are replicated in different ways.3' direction as the fork a dvances. Synthesis and Secretion of Zymogens in Pancreas In an experiment designed to follow the synthesis and secretion of zymogen. In DNA replication. all incorporated radioactivity was found to be present in the rough e ndoplasmic reticulum (ER). a radioactive amino acid was injected into the pancreas of a guinea pig to label p roteins undergoing synthesis (pulse). The other newly synthesized sfrand (lagging strand). Three mi nutes after the pulse. both daughter strands (dashed lines) are synthes ized in their 5" -. Sometime after. The leading strand ts synthesized continuously. 18. but in discreet vesicles near the plasma membrane called zymogen granules. USES OF ISOTOPES A TRACERS IN BIOLOGICAL SCIENCF. radioactivity was found neither in ER. All other proteins. Seventeen minutes after. Thus one could follow the migration of the earlier synthesized radioactive zymogen. Lumen of a duct Rough retlculum Figure 13. Here they are concentrated a nd packaged into the zymogen granules. It appears that these zymogens are synthesized in t he rough ER. The granules then migrate toward the plasma membrane and fuse with it releasing their contents (Figure 13. formed after the first three minutes would be almost non-radioactive. This ensured that only the zymogen synthesized in the first th ree minutes would retain radioactivity. That these vesicles contained zymogen was proved by isolating these radioactive vesicles and chemica lly proving the presence of zymogens within them. Thus with the help of pulse-chase experiment the manner of synthesis and secreti on of zymogens can be ascertained.18). H . most of the radioactivity was found in rough ER which w as near the Golgi complex. 117 minutes later. including zymogens. The synthesis and secretion of zymogens as determined by pulse-cha se labeling.granule complex 518 Bphysical Chemtry After three minutes. These zymogen granules later fuse with the plasma membrane surrounding a duct and release their contents into the duct. large amount of non-radioactive amino acid was injected int o the pancreas (chase). Oufl/ned below are some applications of tracer studies in biological sciences. nor in Golgi bodies. From there the proteins move into the Golgi bodies. and (//) localization of the isotope by detection of radioactivity in dLfferent parts of the system or .owever. only a br/ef treatment is possible. Distribution Studies The study prlmarfly consists of (0 admin/stering a radioisotope to a system or o rgan/sm. owing to the vast number of applications and complexlty of the exper/mental deta ils. 3I ad ministered as NaI has provided much information . or molecular.about thyroid physiology. Such studies can be made quantitative and the time-cou rse of distribution can be determined. ribosomes.g.19). if there is one. or the nucleu s). a whole organ).Illll Isotopes in Biology 519 organism at a later time. (W Malignant tissues tend to have an acc elerated metabolism and hence take up more phosphate than the normal tissues. This can be found by placing a photosensitive emulsion i n contact with" the tissue. Thyroid functions h ave been measured by assaying the uptake of3q by the thyroid gland and the appearance of the isotope in saliva and the urine (Figure 13. The level of detection might be macroscopic (e. microscopic (e. after a particular time more . The malignant tissues will show up as a spot blacker than the normal tissues. Consequentl y ifP is injected intravenously to a patient...g. sub cellular particles such as the mitochondria.2p will be ac cumulated in the tumor. '"" " " fill IIII I I fill IIIIIIIIiiiii ilil lllllill III IIIHIli] tllll IIIIIlillllll IIIII illltllH I'll illii Ifllillllllll IIIII IHIIHHIIII IHI IIIIIIIglill . A wide array of applications of distribution studies can be cited in biological and medicine research: (0 Iodine is an important dement in thyroid physiology. The distribution may be detected at various leve ls of organization. the quantity of the non-radioactive substance in the given mixture may be calculated as shown below.).. More than average distribution of I on the left b'Imtes a normal left thyroid be. Ao = Counts per minute of the tracer added (care must be taken that the amo unt of radioactive cbmpound added (M is very small as compared to the a mount of the non-radioactive isotope in the system). there s no on the r@ht side where the r@ht lobe should have been.19 Thyroid sctntkjram. Thls shows that the rlght ttujrold Isotope Dilution Studies Radioactive isotopes are olten used to estimate the amount of a single substance in a mixture or even the volume of system (e. A° = S. to completely drain the liquid out in order to measure its volume.ilfllHIll IIIII IIIIIIIHIIH IIIIIIImi illii liiIIliliii liIlilI/lliI Sternum Figure 13. M u = Unknown amount of non-radioactive compound in the system. The technique involves (a) introduction of a radioactive isotope of known specific activity into the sy stem.A. blood. cerebrospinal fluid et c. Subject was given an oral dose of 75 mlcrocurs of l 24 hours before. However. From the specifi c activity of the compound isolated and the prior knowledge of the specific activity originally ad ded. and (c) determining the specific activity of the isolated compound. in cases of fluids like blood or lymph. (b) isolating a small amount of this compound from the system after equilibration has been ach ieved.u = Specific activity of the compound in the system after addition ofradi oactlve Mu isotope. . lymph. The technique known as isotope dilution technique is especially useful when it is impossible t o quantitatively separate the given substance from a mixture or.g. However the specific activity of the compound in the mixture will be the same as in the reisolated compound. So.'. A r = Counts per minute of the re-isolated compound.oX Chemtry Mr -.r.r. lr M--. M = Mr orMu = . Bophgs. However. This is so since it has been assumed that the tracer added is of very h igh specific activity and consequently of an insignificant weight as compared to the non-radi oactive counterpart in the system.r or A° = Mu Mr .= S. This is a valid enough assumption since most of the c ompounds of biological interest are avRilable in radioactive form with very high specific ac tivities. in certain cases where compounds of high specific activities are not ea sily obtainable. Mo has be en totally ignored.520 S.A.A.Amount of pure compound reisolated from the system after equilibration wit h the radioactive isotope. S. the amount of radioactive tracer added becomes significant and M° has .A.A.r It will be seen from the above that in all the mathematical relations. " Ar S. = Specific activity of the reisolated compound.A..u = S. . the degree of reduction in the specific activity is directly related to t he amount of non radioactive compound present in the system.A.A.Mo or S.Mu = S.S.Mo S. It is easy to se e that the specific activity of the re-isolated tracer is going to be less than the specifi c activity of the tracer added. Mu = Mr Ao Mo or IVL " Ao Mo .S. the dilution of specific activit y is identical to the dilution of the tracer added.A.Mo + S.r As M0 becomes small.A.r.r Mo Dilution = S.A.A.-S.{2} Ar S.o. S.Mo.o Mo +Mu S. .r ) . the equation (2} will reduce to equation (1}. This decrease will be higher if there is more amount of non-radioactive c ompound present in the system and lower if a small amount of non-radioactive compound is present .O.A.to be taken into consideration.r.. In other words.A. S.r ) --'t. Or. Let us now concern ourselves only with the specific activities.M = S.r.A.o. The equation (1) willthen take a slightly altered form.r.Mo t.---''.A.r.A.A. (tO 45Ca. Transport of long into the tissues in the body has been extensively studied with the help of isotopes. on the other hand. The use of ele ctrolytes. This is an impor tant since ionic fluxes are known to have a profound effect on cellular metabolism. urea and uric aci d with and sodium and potassium pool measurement with Na and 42K respectively. Studies include measuremertt of erythrocytc volume with sCr labeled erythrocytes. Some examples are provided. Transport of electrolytes and of organic substances that are n ormal cell became subject to valid experimental approach because the quantity of tracer substance necessary was so minute that the concentration in the medium was almos t unaltered. measurement of plasma volume with I labeled serum albumin. Membrane Transport Stud/es The classical method for the study of membrane permeability was limited in its a pplication with respect to both the cell types that could be studied and the materials whos e diffusion constants could be determined. 24Na. required such high concentrations in the medium that the possibi lity of altering the permeability of the plasmaimembrane was always present. Thymidine permeability in bacteria was tested using radioactive isotopes 32p and 3H.r There have been many applications of isotope dilution analysisin biochemistry an d medicine. the formation of ATP on the cell surface is the principa l mechanism for the entry of phosphate into the cell interior. (0 Studies with 32p have shown that in the liver. measurement of extracellular space with Br. Availability of radi oactive isotopes changed all this.A. kidney and in the erythrocyte. measure ment of body water with 3I labeled iodo-antipyrene or trltiated water. measurement of total bbdy pools of cholesterol.Isotopes in Biology 521 Mo . Organic non-electrolytes which were not readily m etabolized were the only compounds whose permeability could be studied (such studies were n ot of much use anyway as these compounds were not biologically significant}. S. The . and 4K have been used to study the of these elements through the plasma membrane of various cells. It seems that the bacteria cannot AMP the way they cleave TMP. If so. the radioactivity will be retained on the filter since the cells been retained upon it. thymidine itself is perme able. one can conclude bacteria have broken thymidine to thymine and phosphate and have then taken thes e . The radioac tivity of the double labeled AMP is not retained on the filter. It could also have been that the bacteria were breaki ng thymidine into thymine and phosphate and then taking up both the components. How one test this possibility? . washed and collected by filtration. the radioactivity will be f ound in the In this experiment it was found that radioactivity was retained on the filter. Incubate either a single or double labeled thymidine with bacterial the bacteria are harvested. If thymicine has not entered. After the cells have been purify thymidine and see whether it is labeled.Label thymldine with P and do the above experiment. of Cells and Sub-cellular Particles Isotopes have provided much information about the behaviour. if that is not so and the bacterial ceils show radioactivity due to P. movement. has entered the cell. activitie s and and other functioning particles in the body. For this kind of study it is necess ary .is relatively simple. similar results are not obtained with 2p or 3H labeled AMP. Experiments like these confirm that the bacterial cells actually break the nuc leotide and the membrane. However. Should the above result be taken to mean that thymidine as a whole is permeable to the membrane? Not necessarily. erythrocytes can be cited as an example of this applic ation. it becomes clear that spleen must be directly involve d in removing ageing erythrocytes from circulation. This react/on could conceivably occur with cleavage of either the CO bond or the PO bond.522 Biophysical Chemistry to incorporate a rad/oact/ve element or compound into the cell or particle in su ch a way that it will remain bound for the life or functioning period of the cell or particle. Sampling at the interval of one to three day s permits the determination of radioactive ceils in circulation. Since 5Cr can be recovered from the spleen. One add /t/oval Informat/on that comes to light with this experiment is the fate of the erythrocytes. After labeling the ceils are reintroduced into the blood stream. Me asurement of the/n vWo life span of labeled .D CH2OH . A an exampJe consider the cleavage of glucosel-phosphate as catmfzed by alkal/ne phosphatase. If this reaction is carried out in the presen ce of H2sO enriched water. The half survival time for lo ss of 50% of the labeled cel/s from the c/reulat/on Is used as a measure of the/Ire span. OH + HO . where it is converted to chromic state and becomes bound to the haemoglobin. An aliquot of whole blood drawn from the body is incubated with 5Cr in the chromate state. It remains bound till the death of the erythrocyte. The label moves into the erythrocytes. the former reaction (CO cleavage) path yields glucose containing one atom of CH20H 0 The latter (P --O cleavage) is characterized by phosphate containing one atom of . Isotope Incorporation and lsotope Exchange Studies lotope incorporation studies yield irormation concerning the position of bond br eakage and formation during a reaction. OH During experimentation the label invariably appears in the inorganic phosphate i dentifying P--O bonds as the cleavage site. This enzyme catalyzes the following exchanges. Thus. Sucrose phosphorylase provides an example of the isotope exchange studies. the reaction seems to proceed via a two=step reaction path: . each carried out in the absence o f the second substrate: (I) Glucose-I-P + mPI Glucose-l-rap+ PI (2) Glucose-Fructose + Fructose-4C Glucose-Fructose-C + Fructose These findings are consistent with -the possibility of a glucosyl-enzyme interme diate being formed. Isotope exchange studies yield information about the existence of reactidn intermediates. This is the proof that the hydrogen bonds are n ever permanent and the hydrogen Involved In these bonds is continually exchanged with the hydro gen of the water.Isotopes n Btotogy 523 Glucose-1-P + enzyme Glucosyl-enzyme + P Glucoseenzyme + Fructose Glucose-Fructose + enzyme Both isotope Incorporation and exchange studies have yielded much information ab out the reactions of many metabolic pathways. Biological structures are seldom stationary. carbohydrate degradation. it has become possible to calculate the relative proportion each route. After a certain period o f time a compound . nucleic acid synthesis. Metabolic Studies It can be said without fear of ecaoogeration that the greatest area of applicati on of radioactive Isotopes In biochemlstry has been that of metabolism. A substance. Isolation and identification of metabolltes of major metabolic p athways has been made possible by use of isotopes. to name a few. fatty acid degradation and biosynthesis. proteIn photosynthesis. the TCA cycle. Consider this. and cholesterol and steroid metabolism etc. Moreover. it could be any other biological isotope) is administered to an anlmal. soon the tritium wi ll come to be located In these macromolecules. containing 4C (C is chosen arbltrm-lly. amino acid metabolism. If one incubates these two macromolecules In tritium labeled water. Isotopes have been employe d In the study of almost every phase of metabolism. A. The hydrogen bonds between the two strands of DNA are forming and breaking continuously. So are the hydrogen b6nds of prote In. heine biosynthesis. minor metabolic pathways which have a potential to become major pathways In disease have been elucidated due mainly to the use of pathways exist. In many of the latter. The example is treated diagrammatically in Figure 13. IC. all showing radioactivity du. the process Is Interrupted at various tim es. A beautiful example of the above consideration is afforded by Bl och's experiments on cholesterol biosynthesis. Thus. distribution. Since 14C does not naturally occur in an organism and since it was administered as par t of a compound A. C.20. C. which i s a sort of landmark discovery. where by using 3C. he could establish that all the carbon atoms of cholesterol are derived from acetate. In this way a whole metabolic pathway can be established (the actual experimental set up has many complexities which have been ignored here for the s ake of understanding). The results obtained with doses which are many times as high as the daffy intake may not give an accurate picture of the absorption. and deuterium labeled acetat e. not enough attention has been paid to the desir ability of keeping the total amount of material administered down to the level of the daily intake of the element.524 Biophysical Chemistry E is isolated and purified from the animal and is found to contain radioactivity due to 14C. The exchange reaction s that take place in the bone with respect to calcium and phosphorus have been gleaned from the studies made using gsC and a2p. It can then be surmised that A goes through the steps B.e to IC. He along with his colleagues went on to establish the pathway of cholesterol biosynthesis. it can be said that A is a precursor of E or that E is a product of A. Studies on the metabolism of mineral elements present in large quantity in the b ody have been more successful in general than similar tracer studies of the metabolism of the trace elements. Mineral Metabolism Isotopic tracer technique has yielded much information about the absorption. Extensive us e has been made of this technique to study the metabolism of calcium. distribution and excretion of the mineral constituents of the body. if in the same set of experiment after feeding IC labe/ed A. there is every likelihood that we will be able to isolate intermediate compounds. Metabolic Turnover Time Determination . B. and excretion of the quanti ties the body is normally called upon to handle. by means of radioactivity a precursor product relationship has been established. Now. and D before it forms E. a nd D. it could be found that the proteins of the liver cell have a haft-life of about 5-6 days. . When the same animals were fed the normal or 4N amino acids. In that case too isotopic studies gave the answer. When SN labeled amino acids were fed to animals. Thu s. the previously labeled liver proteins rapidly lost their label.For many years it was erroneously believed that cell proteins remained intact an d stable till such time as the cell lived. liver proteins undergo metabolic turnover. Two different bonds in that reaction could be broken with the s ame products forming. On the other hand proteins of muscle tissue turn over quite sluggishly showing a half-life of about 30 days. In many reactions there is an ambiguity about which bond has actually broken or which has formed. On the basis of these experiments. the label appeared in the polypeptide chains of liver proteins at a fairly high rate even when the total a mount of protein did not change. This concept could be challenged and disproved solely by the use of isotopes. Following is another example where mechanism of enzyme action is laid bare by isotopic studies. The meth od can equally well be applied to carbohydrates.1-phosphate by alkaline ph osphatase as discussed above. lipids. again with no change in the tot al amount of liver protein. Mechanism of Enzyme Action Enzyme action can also be studied using radioactive isotopes. A good ex ample of such an ambiguity is afforded by cleavage of glucose. The obvious conclusion of the experiment was that a relatively high rat e of synthesis of proteins in the liver was balanced by a relatively high rate of degradation. and other cellular constituents. phosphates.and y-phosphates are e xcluded. Fe. If th e same incubation as above is carried out with the four deoxyribonucleotide 5'-triphosphates label ed in the -. °Co radiations given to tumors and many tumors have been known to regress to quite some extent by this In this therapy. More than 100 dif ferent radioisotopes have been used for diagnosis. If DNA polymerase I is incubated with DNA. Among the most important ones are 13 I. in this case the growing chain of DNA. Radioisotopes are very widely used for diagnostic tests.become part of something that is big enough to not to pass through the pores o f the filter. Retention on the filter indicate s that the label has .and later ph otographic gives a clear idea of the area in which the heart muscle is damaged. the 3p remains soluble in trichloroacetic acid and radioactivity is not retained on the filter. Radioisotbpes have been particularly useful in treating cancers. 5Cr is used for blood volume determination and investigatio ns into certain of anemia. II-iodohippuric acid is used for kidney function tests. and all the four deoxyribonu cleotide 5' triphosphates labeled with 32p in the a. Thus. a buffer. P. A sure indication that the P has not become incorporated into the DN A. and 1Xe. A very recent use has been that of °Thallium.Isotopes in Biology 525 Mechanism of DNA polymerization can be understood easily using radioactivity. This may be verified by other experiments. the 32P becomes insoluble in 10% trichloroacetlc acid and can be collected on a membrane filter. This means that Mg+ is essential for polymerization and that all the four triphosphate nucleotides are required to be present in the reaction mixture. a high intensity beam .phosphate which becomes a part of gr)wing DNA and the . radioactivity passes through the filter. Mg2+. Its administration into the patient .position.. (iO Therapy. This isotope mimics potas sium 10n behaviour in its uptake from the blood stream into the normal cells of heart . if the reaction is carried out without Mg+ or with one deoxyribo nucleotide 5'triphosphate less. it is the .Additionally. °F(2. does enter the damaged heart cells. 51Cr. Clinical Applications (0 i. known as the tele-therapy. °SAu. This means that the phosphodiester bond is formed with the a-phosphate of the nucleotides. It however. . or y. Xe i s used for lung function tests. Drugs to be administered by injection and plastic disposables such as and syringes are also sterilized by such treatment.000 curies) from a tiny °Co source is collimated on to the tumor area. it loses its activity by decay very Modern science has reached a stage where it can utilize the energy involved in r adioactive ' for therapeutic purposes. Yittrium-90 has been used for cancer therapy.2 hours. Thus. such implants result tissue. yittrium oxide ceramic beads have also been implanted the pituitary gland. A great ofyittrium implants is the short half-life if the isotope. . Thus. Since yittrium emits chiefly [-particles. heat generated by Pu decay is converted to ele ctricity nuclear powered battery which is used to operate a heart pacemaker. while not affecting the rest of the body. in Sterilization of Foods and Equipments Strong y-emitters are ued to sterilize prepacked food (milk and meat) and surgic al such as syringes and needles. it has effect only in the vicinity. One therefore not have to surgically remove the implant at a later time. The reasoning behind such implants is that since pituitary stimulates a destruction of this gland will slow down the tumor growth also. This pacemak er be implanted in the body and needs replacement once in about ten years. just 64. Tiny yittrium oxide ceramic beads are in a tumor.(~ 1. Alternatively. The technique obviates the need of maintaining an environment. Ab Concentration of test antigen funlabelled ] . It can achieve this even if the mixtu re contains j huge amounts of other materials in which the investigator is not interested. The binding of radioactively labeled antigen (Ag*) to a fixed amo unt of antibody (Ab) Can be partially inhibited by addition of unlabeled antigen (Ag). The exten t of this inhibition Basline Bound antigen Free antigen * 3Ag" 2Ab lag" 2 Ag°. Wit h the development of methods for labeling antigens to a high specific activity.1:2 (c] 2:1 Ratio ' Free : Bound Radioactivity 526 Biophysical Chemistry Idiolmmanoy Radioimmunoassay (RIA) is a highly sophisticated technique and can detect extrem ely small amounts of non-radioactive material. The principle on which the technique is based is deceptively simple and is shown in Figure 13.21 (A & B). very low concentra tions (I0-12 g/ml} can be detected easily. lf 3 mol of radiolabeled Ag (. TtUs ratio can easily be plotted.) are added to 2 mo l of Ab. lt is easy to see that the b of radloactWlty to Ab la tnhlbed by of unlabeled Ag. one mol of Ag will remain free and two will bind to Ab. If the x-axis were a logarithmic scale. A typloal standard curve for radlomumoassay. only 2 mol Ag will bind. However. I mol of each will be bound to Ab.Fure 13. Aga ln. The ratlo of free to bound radloactW in thls case wfll now be 2: l. The ratio of free radloactWi ty to that of bound will be 1:2. 3 mol radlolabeled and 3 mol unlabeled (o) to 2 moI Ab. This ts shown In the upper paneL The boffom panel shows what happens { 6 moI Ag is added. a sIg ht line could be obtWne . the ratio ts golng to vary deperultng upon t he concentratn of the unlabeled antigen added to the rrUxture. because of the equal ¢oncentration of the radlolabeled and unlabeled antjen.21 (A & B) Pritwiple of radloimmunoassay. in other words. ( C). only 50% of the antibody will be f ound binding to Ag*.only 25% of the antibody will associate with Ag*. Dextranoated activated charcoal method. all the antibodies will be found bound to the Ag*. the other 50% would have bound Ag.Isotopes in Biology 527 is a measure of the unlabeled material added. This material has molecular sieving properties (see chapters on chromatography and . Once this standard curve has been obtained. You must have learnt from your chemistry classes that activated charcoal can adsorb many small molecules. Ab-Ag* is collected after the required incub ation period and its radioactivity is measured. If we n ow make the mixture 50% with respect to Ag* by adding Ag. This is done by mixing a fixed amount of Ab and Ag* and placing the mi xture in a set of tubes. The amount oi'radioactlvity so o btained is then plotted against the concentration of Ag (Figure 13. How is Ab-Ag* complex separated from Ag*? Two methods are mainly followed. the amount of Ag in each successive tube is higher . if the medium consists of 10 0% antigen in the radioactive form. Thus.25% Ag* and 75% Ag . This done. a reference curve for Ag must be prepared. and the doub/e-anttbody method. the ra dioactivity in the collected Ab-Ag* is measured for each tube. The value so obtained is fitted in the standard c urve and the amount of Ag is read from the reference curve. However. When the reaction is complete. To measure the concentration of Ag in any given sample. If the medium is made more poor with respect to Ag* .21C). The method consists of covering the activated charcoal with cross-linked dextran . They are the dextran-coated act/vated charcoa/method. the binding for the larger molecules is considerably slower. just the way we prepared standards for spectrophotometric assay. the Ab-Ag* complexis separated from Ag*. one adds an aliquot from theunknown sample to the same Ab-Ag* mixture used to obtain the reference curve. The first one is given below. the other 75% will bind Ag. Even large molecules like large proteins and even larger complexes like the Ab-Ag* complex may bind. To do so. To these tubes a known amount of Ag is added. it is easy to measure Ag in any unkn own sample. CENTRIFUGE =Ag* SUPERNATANT CONTAINS ].=Ag*-Ab ONLY Flgw'e 13.22 The dextran coated charcoal method for separating bound radloactivi ty from unbound radioactive . These large molecules cannot get to the surface of actlvated charco al and will not adsorb. Iodination of proteins is done on the tyrosine or the histidine residues. This is the easiest and a fast way to separate Ag* from Ab-Ag* complex. Quenching. This method cannot distinguish active protein molecules from biolog ically inactive fragments which may still be antigenic. ?-emitters. . Ag* will pellet out while bound radioactivity. Other isotopes such as 4C or 3H are -emitters. unbound r adioactivity. Most of the time the antigen or the hapten used for radioimmunoassay is labeled with 12sI. it has to be seen that iodination of the antigen does not cause it to lose its antigenicity toward the antibody. There is a very good reason for this. Of course. however is sufficiently small to penetrate the pores and is ad sorbed (Figure 13. i. A variant of radio -immunoassay technique. quenching becomes a severe problem. Since the mixture containing the antigen contains many p roteins. i. then it is conjugated with one of these resid ues and iodination of the conjugate is carried out. Radioimmunoassay is a wonderful diagnostic technique and yet it suffers from one shortcoming. The pores here are sufficiently small to preclude entrance of either Ab or AbAg* complex. prostaglandins. -emitters have to be measured in liquid scintillation counters. The power of the technique has made it t he one of choice in determining the concentration of protein hormones.528 Bophysical Chemistry electrophoresis). If the protein does not contain tyrosine or histidine. does not remain a p roblem with.e. and even such smaller molecules as steroids.e: Ab-Ag* will remain in the su pernatant. 12sI is a strong ?-emitter. the immunoradiometric assay (IRA). as such. Immunoradiometry There are cases where a radioactive antigen is not available. Labl|ng the antigen. can be adopted in such cases. There is no such problem with I since it is a Y-emitter and consequently radiates higher energy. carcinoembryonic an tigen. Thus if dextran covered charcoal is added to a mixture of free Ag* and A b-Ag* complex. Ag* will bind and Ab-Ag* will not. hepatitis B antigen. and morp hine related drugs.22). If the mixture is then centrifuged. Free Ag*. This is added to the column of antigen adsorbed on cellulose or sepha rose. This mixture is now added to the cellulose/sepharose to which Ag has been bound. ecological studie. The principle of immunoradiomet ric assay is described diagrammatically in Figure 13. The antigen cannot be labeled for a variety of rea sons. which has already reacted is automatically eluted. and sediment s.23. Radioactivity in the eluent can be counted and will be a measure of the antigen in the given sample. The coupled antibod y is now iodinated with 51. The antibody specific for the antigen will bind to the antigen and will become adsor bed. Ab*. To label the antibody. So.The logic here is as follows. Among other IgG. radio dating of rocks. the antigen specific for it is adsorb ed or coupled to a stable substance such as cellulose or sepharose. fossils. Anti-sera raised for the antige n in a suitable animal is obtained. This labeled antibody.s involving migratory and behaviour patterns of animals. Subsequently. Other . is now used for assay of t he antigen. this also contains the antibody specific fo r the antigen concerned. The Ab*. an excess of Ab* is mixed with the sample containing the antigen. Subsequent washing of the column will remove the unadsorbed antibodies. For the assay. label the antibody. . The Ab* which has not reacted with Ag can only become bound to Ag in the cellulose column. it is eluted from the column by changing the co nditions of the wash medium sufficiently. Radioisotopes are used in many other areas such as pharmacological studi es involving development of new drugs. 131 in a suita ble mixture so that tyrosine residues pick up the iodine. Now denature another aliquot of the protein and subject this too to iodinat ion presence of radioactive iodine 131. React the protein with radioactive iodine . Count the precipitate. that tyrosine is the only amino acid that can be iodinated readily. Suppose that the count given is cpm. .Iodir/ate 529 Isotopes in Biology ------. Students will recall from their studies on the thyroid pri nciples. Just how this app lication works is being illustrated with the help of an example. After lodination is over. precipitate the pr otein count for radioactivity.Antigen Cellulose IgG Incubate to adsorb Specific antibodies Add Add Cellulose ' antigen antigen Count Radioactivity Figure 13. Suppose now that the count is 9000 cpm.23 Principle of immunoradiomery Study of Protein $tractre Most studies on protein structure using radioisotopes are based on iodination of tyrosine in the peptide chain. Suppose that sequence analysis of a protein has provided the data that the given protein residues of tyrosine. After this precipitate the protein the help of trichloroacetic acid. These same tyrosines exposed upon denaturation and therefore the count given by the denatured protein was .The above data tells you that the native protein picked up less iodine than the denatured This goes to show that some tyrosines must have been buried in the three dimensi onal in a hydrophobic environment unavailable to the iodination. however. readily available. SOME INFORMATION ABOUT COMMONLY USED ISOTOPES Deuterium. This readily available isotope. Nitrogen-15 and Oxygen-18. Car bon. It is possible to grow quite large organisms on heavy water (D=0). Carbon-14. other four were buried in "the three dimensional conformation. [4000/9000] × 7 = 3. or the scintillation counter. is probably the most important and the most extensively used in biology. it is relatively safe if t . The famous Messelson and Stahl experiment made use ofSN to pro vide evidence that the DNA replication might be semi-conservative. Thus 3 tyrosines were available for iodination and out of total of seven. Although it has a fairly long half-life. It has been incorporated into a number of important biomolecules and most of them are available on the market.530 Ba>physa:al Chemtry A small Calculation will also give you just how many tyrosines were buri ed. This is very cheap. It should. These stable isotopes. Studies of the results of such substitutions have been very informative about numerous m olecular processes. This isotope emits a very low energy -ray having a maximum energy of 18 kilovolts. It can be readily detected by the photographic emulsion. a Geiger cou nter. It has been extensively used in the form of tritiated thy midine as a means of specifically labeling DNA and has provided much useful information in molecul ar biology. It requires some care in counting because the beta rays travel just about 15 cm in air. and can be measured by the fal ling drop method very simply. be understood that suc h a compound constitutes a very serious hazard because it is especially geared to enter and s o affect the genetic part of the cells. which are obviously intimately related to biological compounds. with a long half-life of 5000 years. Tritium. are reasonably easily available. Bloch made use of3C to prov ide evidence that all carbon atoms of cholesterol are derived from acetyl CoA. and a single beta-ray of maximum energy 130 kilovolts. Thus any thickness in the sample is de trimental to measurement. and nitrogen are available up to 96% concentration. It has also been tried as a cancer cell killer (the cancer ceils replicate much fas ter than the normal cells and therefore take up more tritiated thymidine injected in the system as c ompared to the normal ceils leading to their death). Carbon-13. and so can be used in studies which need very heavily labeled material. aken internally. More or less of equal importance with carbon. It has been used to st udy sodium transport across plasma membranes. About 250 microcuries are known to be tolerated well. it is therefore necessary to use it immediately on acquisition. 1.detect. Ne arly every protetn contains one or more of the sulphur containing amino acids {methlontne. Since sodium readily ionizes it excha nges very rapidly. This quite short-lived. it t oo short to store it commercially. this element. Thus. This same half-life however.6 MeV makes it easy to . It can be produced by bombarding sulphur with neutrons of over 1MeV energy. With more or less similar characteristics as 14C (the half-life of o nly 80 days is an exception). . It can be used wherever a study of sodium is needed. It has no gamma radiation and consequently can be handled in relatively large quantities without danger. 5S has been used as an important tracer in protein studies.4 and 2.8 MeV) is readily ava ilable. Sulphur-35. extremely active element (half-life i5 haur. cystetne. Its maximum -particle energy of 1. The half-life of 15 days makes it possible to employ it in relat ively long biochemical processes (elucidation of metabolic pathways). Phosphorus-32.4 MeV beta-particle energy + two gamma rays of energy (1. studies of longer duration (such as a determination of pathway of a particular metabolite) are not normally possible with. Sodium-24. The resulting P is tota lly separable from sulphur giving rise to very high specific activity 3=P.14 is P (others hav e called it "almost the perfect /sotope to use"). These events can also take place when radiation interacts with living tissues. two partlcle energies of 0. This is precisely the mechanism by which radiation can have dele terious effects on life {see Box 13. . decaying by electron cap ture).1 and 1. Iron. Extensive use has been made of both the isotopes in many studies involving blood and its formation. Two forms are avzilable: Fe (half-life 2:9 years. Although every individual in the world Is exposed to a s mall amount of radiation arising from natural and man-made sources.46 and 0. S has been recently widely used in DNA sequencing studies. Fe emitS mainly X-rays and is therefore hard to detect. We have discussed that Iorflzation and excitation are the two events that take p lace when radiation interacts with matter.3 MeV). of A is initially higher than the specific .7). It Is therefore useful for a biologist to know what are the tolerable radiation doses and to limit his/her exposure to those levels or level s even lower. exposure to radiation Is of more than mundane interest to a biologist who Is exposed to radiations from tracer experim ents which have become so common and useful. and bFe (half-llfe 45 days.26 MeV + y-rays of e nergy 1.A isotopes tn 531 cystlne) and thus can be labeled with S. One should always wear disposable gloves because radioactive compounds can be ab sorbed through the skin. There are.1 rem per week). a unit of mu ch better use for biological exposure has been introduced and is known as the rein. The different radiations are assigned what is known as qualltyfactor (OF). a dummy run before actually using iso tope is advisable. alpha particles are about 10-20 times more damaging to the tissues as compared to the beta. The experimental set up and the worker should be separated by a Perspex shield b etween them.. Thus. One rad equals 100 ergs per gram of material. hands and forearms.and gamma -radiations have been assigned a OF of I while alpha. It is a measure of the energy liberated in a specific material by exposure to ra diation. the beta. The rem is defined as the dose in rads multiplied by the quality factor. The usefulness of the rem lles in the fact that it takes into account the varying effect of different radi ations into account. other tissues which are not so sensitive. which are most involved in exposure while carrying out biolo gical experiments.radiation has been assigned a OF of 10. The stocks. The time . partlcularly if they are gamm a emitters. Precautions Certain precautions should be rellgiously followed when working with radioactive isotopes. should be shielded in lead shielding. One such unit in use is the rad. The whole.spent manipulating stock isotope solutions should be minimized. however.and gamma-radiations. If the experimental mix containing the rad . allowing only the hands to be exposed. This whole body dose has been set up considering the m ore sensitive tissues such as the gonads and the bone marrow (as a general rule fast dividing tissues are more sensitive to radiation). If the protocol is thought to be complicated. The tolerable exposure limit for th ese organs is set up at 75 rem per year and up to 20 rem in any 13-week period. 0.(alpha-particles have little penetrating abili ty.e.532 Biophysical Chemistry Let us define units by which we can quantitate exposure. For example. can tolerate a higher exposure. and therefore their effects become concentrated in the small area to which they are limited). Although rad is quite useful.body tolerable dose of radiations has been set at a maximum of 5 rein per year (/. Its size makes it easy to carry. . it should be properly shielded. One may also carry apocket dos/meter. The badge holds a photographic film which when developed at a re gular interval can provide an estimate of the exposure by the degree of blackening of the film. They can wear af/ /m badge on their lapel. The radiation workers should monitor their exposure radiation. it is roughly of the size of a fountain pen.ioisotope is to be transferred to other parts of the laboratory. This instrument is a miniature electroscope: Its scale is directly calibrated in dose units and therefore one can determine the dose of exposure at any given time. wear with radio-isotopes. The liquid radioa . it becomesvery dangerous because in this form it will be incorporated in the gen ome and will continue to send radiation in the tissue for a very long time.Isotopes in Biology 533 Accidental Ingestion of Radio-Isotopes We have so far discussed the external exposure ordy. always use oke in a radioactive laboratory. drink. can be taken to avoid ingestion include (t) to always. This term is much more useful and can provide a correct index of the danger of an isotope if ingested. while ingested radioactive sodium will be distributed throughout the body more or less evenly. Radio-isotopes are much-mor e daungerous internally. other isotop es such as 3I. Obviously.will not be so dangerous because it will be rapidly excreted by the lungs as 4CO2. the isotopes having short biological haif-llves will be less dangerous than the ones having a long biological half-li fe. (//) to never to mouth-pipette a mix c the safety pipette. On the other hand. Different isotope s can have widely varying half-lives ranging from hours to several years. The isotopes which are accumulated in a particular organ are much more dangerous tha n those which are distributed evenly. if 4C is ingested in t he form of "C-thymidine. and 3p will accumulate primarily in the thyroid (3I) and the bones (4Ca an dP). Various factors have to be considered in assessing the danger from i nternal radiation from a given isotope. and (/v) ly. some basic aspects will be discussed. 43Ca. The values for radioactive half-life and the biological half-life can be combined to arrive at a term which is known as the effective half-life. The space does not permit us to go into the details of the se factors. Thus. Thus C ingested in the form of HCO. The biological half-life (time taken for half of a given amount of radioisotope to be excreted from the body) of different radio-isotopes is one such factor. (iff) to not to eat. and even sm to carry out radioactive work in designated laboratories on The disposal of radioactive material should also be done with due care. containe rs to store solid radioactive waste are provided in every designated laboratory. The precautions that disposable gloves while working ontaining radio isotopes. however. The soli d radioactive waste requires special storage before it is disposed off (incinerated). Different isotopes are distributed differently in the body. The form in which the radio-isotope has been ingested also determines the extent of danger. DOSIMETRY The techniques given below are used for the measurement of dose and dose rate to which biological systems are exposed. . (i) The ionization chamber.hv = Fe3 + eThe concentration of iron (Ill) so produced can now be determined in a spectro-p hotometer at 304 nm. The same principle is used here. It is based on the prin ciple that the energy of radiation (hv) can knock out an electron from Fe2÷ converting it to Fe3+ in aqueous solutions. This dosimeter is mostly used for monitoring personal exposure by . It uses the of photographic film induced by ionizing radiations as its principle (see sectio n on methods). This is a chemical dosimeter. This is perhaps the most widely used type of dosimeter. Fe2 . We have already discussed the principle of ionizatio n chamber. This system has a good tissue equivalence and can be applied over a l arge dose range.ctive waste can. The only change is that the ionization chamber is calbrated so as to give the measure of radioactivity directly in dose units. be suitably diluted and put down the sink. however. (ii) The Fricke dosimeter. (iii) Film dosimeter. one can measure the exposure by the extent of darkening of the.693 6. They are based on colorimetrlc measurements. (iv} Thermluminescence doimetry. As the number of light flashes (excitons) is proportional to dose. (a)) .14xi02 days[) = 2. {v) Other t. The system is also usefhl over a very large dose range. Mn4 has a half-llfe of 314 days.21x 10-days-] 314 days x 24 hr/dayx 60 min/hr x 60 sec/min .and sec-]m and (b) percent of initial radioactivity remaining in a s ample after 80 days. Certain crystals (e. Calculate (a} the decay constant in ter ms of days. This method has good sensitivit y.. At a later time when the film is developed.693 534 Biophysicd Chemistry workers in the lab. the trapped electrons return tO the ground state emittingll ght in the process.film.l'ence and can be made very small usually by mounting them on teflo n. 0. Severa/other techniques of dosimetry are availa ble.hniques ofdoslmetry. however. When such crystals are heated.314days 3. discoloration of crystals.0. CaF2) have the ability to store radiation-generated electrons In crystal defects over a very long period of time .g. are not used frequently. SOLVED PROBLEMS 1. the amount of light emitted gives a direct measure of the dose tO which one is exposed. it is worn usually on the coat lapels and as a ring on the l ingers wht/e working with radioisotopes.tu2 . These dosimeters hav e excellent tissue equiv. mortality rate of bacteria on exposure to ionizing radiation.693 0. But careful standardization is essential. as the results depend strongly on the emulsion pro perties and on the procedure of development. and conductivity alternation in semiconductors. turbidit y formation on glasses or organic polymers. These systems.93x I0 Arts. 0.14x101 (8.1xi0--'.693 0.6xI No 2.3 = I0-012.21 x10-3)[80) = O.693 314daysx86400sec/day 3.923 Cesium .3 x 10-2 sec - 27.N0 = 100% 2. 1768 N 1o.0. Calculate the fraction of ceslum .0.log N = 0.= )t N let.3 log = -.100.077 or log N 2. .64x10') 69.or [ ) =2.1768 0 077 log 100 .137 that decays (a) per year [b) per minute.000 .077 = 1.137 has a half-life of 33 years. 5256 × I0 1 0. 2. 1 = 0.2 x I0-) (3.3 x 10 This effectively means that 2.1× 10-2 yr.1 x 10-2 atoms per atom decays in a time period of one year.25 x 108 radioactive atoms decays per year.2.535 0.0x 10-a rain (b) = (365) (24) (60) 0.1xi0-2 yr-I = = 4.62 2.25xi0s or 4. 1 atom out of 112.atm Number of K° atoms = .023 x 1023.35% x 75 x 10ag = (1.012% of the potassium in nature.atms/g . (a) Calculating the decay constant 0.15 x 10-2 g 3.693 6. K° (ta = 1.93x 10 yr-1 =2.15 x 10-2 g x 6.693 Isotopes in Biology A.4762x 102 = 47.1 x 10-2 Thus one out of every 47.1×10-2 yr33 yrs 3. Or. To.al K° = 0.5 x 10-3) (7.0 x 10-a Thus one out of every 0.35% potassium by weight. The man body contains about 0.3 x 109 yr) consUtutes 0.012% x 0.1 x 10-2 atoms decay per year.5 x 10) = 3.62 radioactive atoms decay per year. Calculate the total radioactivity from K° decay in a 75 kg human. 74x 102°atoms 1. it takes a long time to decay after the death of the animal. ¸ -dN .3x109 x365x24x60 =6. Since C* has half-life of 5700 years. Given t hat the decay constant of C is 2.93 x I0-14 DPM = = kN = (1.74 x 1020) dt = 4.40 g/g.014x10-Is min-i 6.014 x I0-) (4.81 x 108 DPM Scientific data shows that the carbon compounds being synthesized currently by l iving systems contain enough C to give 13 DPM per gram carbon. and (b) The age of a well-preserved animal which is emitting 1 dpnYg atom.31 x 10-1° min-L can you calculate? (a} the abundance of C of the carbon in living systems.atom = 4.93"× 10-min = 1. (a) dpm/g = = N dt . x 525600 = 1.3 log 13 = 1. t = -1.1139}= 1.216x101. {a} Calculate the specific activity of pure C4 in terms of dpm/g.31×10-1° 1 gram of carbon contains 1 g x6.atom = 5.023 x 1023 atoms/g .216x10--or t=2. of C4 atoms per gram carbon and .at(m 12g/g .x (365) x (24) x (60) = 2.536 Bohsca Cheratstry Where N = No.216 x 10.12x 10-w% 5. as given is 2.02 x 1022 atoms Carbon Decay constant is given in terms of min-I.56 .13 = 2.-.3i x 10-1° N 13 N = = 5. abundance = x 100°/o = 1.31og13 =)t 1 or 2. 5.63 x 10I° atoms C141g carbon 2.31 x 10-1° rain.1139) 2.31 x 10-I° min. .216 x 10-t = (2.63 x 10I° atoms of C14 .3)(1. Converting it to yr-I.31 x 10-°min.yrNow 2. 2.1216 x 10:t {2..3)(1.1 x 104= 21000yr.02 x 1022 total atoms of Carbon. 31 x 10-° rain Ig I g CI" = 14g/g-atom = 0.(b) What should be the maximum specific activity {ci/mole) at which uniforml y labeled L . (b) Since the term of specific act/vity here is in Ci/mole.31 x I0-° rain..0714 g-atom N = 0.. Specific activity = 9. AN g dt N means the number of atoms in Ig oft.023 x I02a atoms/g-atom N = 4. let us convert t he dpm term of the previous problem into Ci.3 x 1022 atoms dpm -dN .leucine .94x lO2dpm/g = 4..48 C/g 2.x 4.0714 g-atom x 6.2 x 1012dprn/Ci . 9.C14 which has specific activity of 150 mCi/mmole? Given that the decay constant ofC14 is 2.0 leucine . dpm = 2.94 x 102 dprrdg.3 x 1022 atoms.CI may be prepared? {c) What proportion of molecule will actually be labeled in a preparation of L . (c) Also determine the specific activity of pure chrom ium .9 (c) 376.023 x 103 atoms. Given that the half-life of chromium.22 × 10I dpm -dN dpm = = XN dt . of chro mium in 1 Ci of pure chromium-51.Isotopes in Biology 4.28 x 1017 radioactive atoms/Ci.73 × 10-5 N 2. .-.2Ci/Mole .labeled molecules = 39.48 Ci/g x 14 g/g-atom = 62.'. (a) 1 Ci z 2.51.8 days and the decay constant per minute is 1. 150 Ci/mole × 100 = 39. 2. .9% Calculate {a) the number of radioactive atoms and (b) the weight in gms.'.2 Ci/mole.28 x 10v 1.1. the weight of 1. % of C14 . (b) 1 g-atom of chromium-51 (51 grams) contains 6.7 Ci/g-atom = 376. maximum specific activity = 6 g-atoms]mole x 62.22 × 10I = 1.51 is 27.'.22 × 1012 N = . Chromium .28 × atoms should be .51 contains 1.'.7 Ci/g-atom L-leucine has 6 g-atoms of carbon per mole.73 × 10-5 rain-I.73×10-5 . 94×10-6g Weight in gm.atom or.1. 4.023 × 1023 X 51g = 10.2 Ci/mg A small tube given to you contains I m Ci of L-histidine-C14 in 3 ml aqueous sol ution. 1Ci = 0.703 x 106 '.28 × 1017 atoms/Ci.atom Thus the specific activity of pure chromium-51 is 92.781 x 10-Iv Ci/atom× 6.781× 10-]7 Ci/atom or 1.28× 10v atoms 0. .(a) the concentration of histidine in the solution. of chromium in 1 Ci pure Chromium-51 is 10.CiJg .84 × 10-6m. (c) Pure chromium-51 contains 1. The specific activity of the amino acid is 200 mCi/m mole. Find out -.703 x 106 CiJg-atom. = 92. and (b) th e activity of the solution in terms of CPM/ml if the counting efficiency is 65%. The amino acid is uniformly labeled.2 Ci/mg 5 I000 mg/g .023 × 10 atoms/g-atom = 4.28 × 1017 6. '. mCi in 3 ml.'. Activity per ml should be 1.005 mmolelmCi 200 mCi The tube contains 1. 1 mCi = 2.22 x 1012 dpm .2.22 x 10 dpm .443 x 109 CPM But then this is the activity in 3 ml. the number of mmoles that correspond to lmmole lmCi - = 0.005 mmole ". Thus the concentration of.65) CPM = 1.(b) Biophysical Chemistry The problem states that 200 mCi is equivalent to 1 inmole.0 x 10s dpm/] moJe.0mi .0ml = 1.'. = 0.001667mmole/ml 3.22 x 109) (0.443 × 109 CPM = 0"481xl09CPM/mlor 4. total activity at 65% counting efficiency should be (2.81 x 109 CPM/ml 3. 1 Ci ---." mmole/ml.81 x I0 CPM 8. Activity per ml = 4.667 x 10-" mmole/ml.L-histidine in the tube is 1. 0.667 x 10-. How will you prepare 100 ml solution of a 10-2 M solution of glucose (1C14) so that the monosaccharlde has a specific activity of 2. You have been pr ovided . and £ractionated for purification of L-cysteine. Colicells were grown on a defined medium which contained S3 sodium sulphite a s the sole source of sulphur.) The amount of radioactivity needed will be 10-2 M = 10 I moles/ml. The specific activity of radioactive sulphur was 4. Now.0 x 106 dpnY/ mole = 2. 10 I moles/ml x 100 ml = 1000 l moles. let us find out how much radioactive stock solution wig be needed to provid e 2.0 x I0 dpm WIll be required to prepare I00 ml of the required solution with th e required specific activity. 1 mCYml x 2. the required solution wig be obtaine d.91ral 2.0 x 109 dpm. 1 M solution of unlabeled glucose along with a stock solution of ucose (1.22 x ]09 dpm/ml 2"0xI0dpm = 0. E.22x 109 dpm/ml . The ceils were allowed to grow for several generations at the end of which theywere harvested.22 x lO dpm/mCi = 2. I0 gm.-.C1 4] (40 mCYmmole and 1 mCi/ml. wet weight of ceils was used for . Purifi ed L-cysteine gave a count of 160000 CPM. If 0. lysed.O. 1000 moles x 2.39 x 104 CPM/ mole.91 ml of radioactive glucose solution is added to 1000 I moles of unlab eled glucose and the solution diluted to 100 ml.0 x 109 dpm 2. On the basis of the above. The medium was so nazxipulated that further growth was improbable. The following table depicts the specific activity of phos phatidylinositol at dllferent tlme periods.5rag protein . 11.I0.'. Ir.I.5 mg protein.000 CPIV mole . The specific activity of the enzyme is 0. it could be determin ed that initial rate of radioactivity incorporated into glycogen primer was 3600 CPM/min . calc ulate the rate of enzyme reaction in terms of l moles per mg protein per minute.32 units/mg protein. After several generations. B. Fracti onation procedures were performed.C4.'. the cells were harvested and resuspended in a fresh medium containing unlabeled inorganic phosphate as the source of phosphoru s.157 I mole/min (b) u = = 0. (a) The rate of enzyme reaction can be calculated as follows: 3600 CPMimin = = 1. subtl/is culture was grown in a defined medium with P as the only sou rce of phosphorus. A cell free extract capable ofsyntheslzlng amylose was incubated with gl ycogen primer and radioactive Glucose.157 I mole/mln 0. for phosphatldyllnositol which was assayed for quanti ty as well as radioactivity. What is the speci fic activity of the enzyme? Am.. Rate of enzyme reaction is 0. calculate the rate of turnov .terrupting the experiment at various tlme intezvals and assaying the radioactivity incorporated.57x10-] t mole/min 23. l.om this cul ture aliquots were drawn at regular time intervals and their lipids extracted.321 mole/rag proteinJmin 0. can you calculate (a) The rate of enzyme reaction in terms of I moles glucose incorporated per min ute? (b) If it is given that the reaction mLx-ture contains 0. From the data presented.phosphate . The radioactive isotope was uniforml y labeled and gave a specific activity of 23000 CPM] mole. .linositol (half-llfe} in B.er of phosphatidy. subtf/fa. more than half of the pa2 at 30 min has disappeared.442 19. However between 30 min and 120 min.744 From the data itself a rough idea of half-life of phosphatidylinositol can be ha d.3 log ffi K (90rain) 14.540 Time elapsed after resuspension (mu} Specific activity of phosphatidylinositol (CPM/.347 3.977 11. First calculating the first-order rate constant.456 34. K: 34.000 45. t/2 should lle somewhere between 60 and 120 rain. 442 2. In the first hour less than half the label has been removed as evident from the cou nt.771 14.977 . mole P) 0 30 60 120 150 180 300 60. .7 x 10 CPM/mg or 1. .as NaSO in the system? A.08 gm.7 x 1 0' CPM/mg x 233. Half-life of phosphatidylinositol in B.A.893 = 0. Mu = Ao S. What is the amount of non-radioactive SO42. the specific activity of reisolated BaSO is 1. subt///s is 75 min.0256)or K = 9.r.6 mmoles u 3. Now.'. . It is found that the aliquot gives a count o f 1. M.6 m moles x 142 mg/m moles = 1079 mg or 1. 12.299 = {0.24 x 10-3/min 0.(The numerator and denominator are counts removed by 90 rain) K = 2"3 Iog2. = 7.'.97 × 106 CPM/mmole But we have to express the amount of SO .5 mg mmole = 3.7 x 10 CPM per mg of BaS3rD. M = = 7. To calculate the amount of NaSO in a given system.075x 103 5 min t12 ' = K 9. S.24xI0-3 .97x 106 CPM/m mole. The label is found in Has.to the system.r 3 × 107 CPM . the researcher adds 3 x 10 CPM of carrier-free SO . A small aliquot is drawn from the sample post e quillbration.08 gm Thus the amount of NaSO4in the system is 1.'.in the system as NaSO.693 0. . No wonder he is selling at such a low price.) =l-xl .1 gm %. M.2 × 104 CPMImg) were added to 50 mls of heparin solutio n supplied by the manufacturer. Determine the dpm of C and Ha. 14. This means that th e manufacturer is supplying 0.000 CP M in channel 2 of the scintillation counter.8 x 104 CPM/mg. it is clear that Ha contributes littl e to the counts in channel 2.labels has been chromatographically isolated from the cytosol of E.1 grn % heparln solution at half the price of other suppliers.Isotopes in Biology 541 13. 10 mgs.Omg.5 90000 . A commercial manufacturer of heparin solutions is claiming that he is se lling 0. (S.000 dp m gives 8000 CPM in channel 1 and 20 CPM in channel 2.and Ha . .000 dpm gives 6000 C in channel 1 and 12.0 1)10mg . in the isolated compound.000 CPM in channel 2 is due to C' alone.1 10mg = M = 30. of C'4 labeled heparin sulfate (7. heparin. :S--r. . The compound gives 50.Ao lMo (7"2x104 ) (4.coli. Therefore it may be safely assumed that all 90. An Ha standard having 40. A small amount of heparin was reisolated from the solution and it was found to have a specific activity of 1.Find out whet her the manufacturer's claim is true. From the measurement of standards.000 CPM in channel 2. To check his claim.0. 000 CPM in channel 1 and 90. Again from the standard measurements it is clear that the efficiency of channel 2 for counting C4 islust 50% [ -. A C'4 standard known to have 24. A substance containing C'4. Background counts have alre ady been subtracted from the data.06 gm % solution of heparin while claiming 0. " Thus 50 ml of the solution provided contains 30 rag. B & C as a function of time.20) (Ha dpm) .C' and H3.25) (180000) + (0. Two compounds.25)(18oooo) 5OOO 0.5 . B & C...'. dpm due to Ha = 25000 dpm. Compound A labeled with C4 was introduced in a perfused liver and the appearance of label in other compounds was followed. Given below are the speci fic activity charts of A. 50000 = (0.180000 The 50000 CPM of channel I are due to contribution from both .2 .'.000 CPM of channe l 1 represents 25% of the C'" dpm and 20% of Ha dpm.5 can you determine from the charts whether B or C is the immediate produ ct of A? .'. Taking assistance of the rule s given in Box 13.2 0. were found to pick up the label. counting of standards in channel I tells us that the 50. .. Again. (5oooo)-(o. The dpm of C4 in the given compound is 0. J.. Butterworths..Time (a) (b) Fig. 3. Isotopes and Radiation in Biology... (Reason it out) Suggest/ons For Further Read/rig 1. C & D as a function of time.C -. D.B -.. Acad em/c Press. .. 1972. On the basis of the fo-ll owing chart of specific activ/ties of A. ed. Isotopic Tracers in Bbchemistry and Physiology. A. 4.) 16. On the basis of elementary studies a group of scientists has proposed th at a path way A . can you det-ermine whether the surmise of the scientists is true? If you have reasons to believe that the pathw ay should be different from the one suggested. 2. Horrocks. L. please state those reasons along with the a ltered pathway that you believe is more apt to occur. Sacks. Specific activity of A and two potential products (a) B & (b) C Ans. 1972. C is the product of A. Thornburn. The pathway should be A -. C.542 ure Biophysical Chemistry Time -. 1974. Dyer. McGraw-HI11.D operates in the cytosol of most cells. 1953. Heyden. (Reason it out. C.D --> C --> B. B. ed. Arts. Liquid Scintillation Counting. Application oflAquid Scintillation Counting. You are also told that the specific activity of the labeled compound is 250 mCi/ m mole.0 x I0s DPM/m mole? Yot are provided a 0. How will you prepare a 50 ml 10-3 M solution of L-cysteine -S3s in which the amino acid has a specific activity of 2. (c) 9.0 ml solution. Can you calculate (a) the decay constant [ .6 x 10Is atoms/Ci.25 x 10S/day {b} 63. An.1 M solution o f unlabeled . (a) 4. (b) the weight in gms.1% 2.32 x 10g radioactive atoms decays/rain. (a) 6. C4 has a half-life of 5700 years.51 x I0-s gm. 3.C4 (uniformly label ed) in 4.) per day & (b) the percent of original radioactivity remaining in a given sample after 90 days. Can you calculate (a} the number of radioactive atoms in 1 Ci of pure P. Calculat.Isotopes in Bbology 543 1.78 x 10s CPM/ml 5. Half-life of Ca4 is 163 days. of phosphorus in I Ci pure pa2 & (c) the specific activity of pure pa2? An. You are given a vlal containing 1 mCi of L-Serine -. An. at a counting efficiency of 50%. Arts. (a) I x 107M (b) 2.e |a) the concentration of L-serlne & (b) the activity of the solution in terms of CPM/ml. (a) One atom per 8225 radioactive atoms decays/year.3 days. (b} One atom per 4. 4. Can you calculate the fraction of C4 a toms that decays (a} per year (b) per minute. (b} 3.125 x 10s Cl/g-atom. The half-life of P is 14. B.sodium s ulphite as the only source of sulphur.4 x 10s CPM/m mole. The initial concentration of. The total volume of the system did not exceed 1.5 ml. Ss. Find out the intracellular concentrat ion of ° L-methionine in the organism. 2. it contained enzymes required for methionine biosynthesis. {a) in term of m moles/rain.Ss An. Calcu late the rate of enzymatic reaction. A cell free extract which contained 720 mg protein was mixed with C4 labeled met hylmercaptan of specific activity 2.An. 6.L-cysteine & a stock solution of L-cysteine . extracted & fractionated for L-methionine. The fraction co ntained 53000 CPM of S35 per gm wet weight of the cells. Since the cell-free extrac t was from a microorganism. Every ml of the medium contained 2 x I0s CPM of radioactivity. . given that 1 gm wet weight corresponds to 0. (45 m Ci/mmole & I mCi/ml}. the cells were harvested.2 gm dry weight & 0. After a few days. megather/um was cultured in a defined medium containing Ss . It could be dete rmined that the rate of incorporation of radioactivity in methlonine was 2240 CPM/min.31 x loS M. sodium sulphite in the medium was 0. O-acetyl homoserine was als o added. & . Aliquo ts were drawn and fractionated for L-methionine at different intervals.007 M.8 ml intracellular water. Biophysical Chemistry (b) 1. The table below gives the specifi c activities of phosphatidylglycerol at different time periods.33 x lOm moles/min. From this culture aliquots were drawn at regular time intervals and their lipids exacted.3 x 104 p. Further growth of cel ls was disallowed. moles/mg proteirYmin. Time after resuspension (min) Specific activity of phosphatidyl glycerol {CPM/mmole P) 0 30 6O 90 120 150 63261 42113 27996 18763 12502 8302 . (a} 9. Fractionation' procedures were performed for phosphatidylglycerol which was assayed for quantity as well as radioactivity. coli cells were grown on a defined medium with only Pia2 as the sole source o f phosphorus. E. coll. calcula te the rate of turnover of phosphatidyl glycerol (half-life) in E.544 xs. From the data presented. After a few generations the cells were harvested and resuspended in a fresh medi um containing unlabeled inorganic phosphate as the source of phosphorus. 3 min.Ans. Calculate (a) Ha d pm & (b) C4 dpm in the sample.4 x 104 CPM/mg) were dded to a c ommercial solution of insulin containing an unknown quantity of the hormone. Historical specimen can be dated with the help of radioactivity. A small amount of insu lin was then isolated from the solutionand it gave a specific activity of 0. An$. Which of the following according to you will disintegrate faster .labeled insulin (2.7 x 104CPM/m g. Calculate the amount of unlabeled insulin in the solution. 10.deuterium or tritium . Milligrams of C14 -.'P or deu terium? 15. t% = 51. If a particular isotope preparation is said to be 2 Ci. Why do you use ass and not a2p to label nucleic acids? 12. (a) 12500 dpm (b) 90000 dpm. 11.adenine.Uracil yielde d 25000 CPM in channel 1 & 45000 CPM in channel 2 of a dual-channel scintillation counter. 16. A C1 4 standard containing 40000 dpm yielded 10000 CPM in channel 1 & 20000 CPM in channel 2. & Ha -. You are to study kinetic parameters of nucleotide incorporation in a gro wing nucleic acid chain. Why do historians prefer 14C dating?. 2119 mg. Which of the hydrogen isotopes . So how do you label nucleic acids with asS? 13.will you use and why?. An Ha standard containing 200000 dpm yielded 40000 CPM in channel 1 and 100 CPM in cha nnel 2. Sulfur is not found in nucleic acids. Background correction has already been taken into account. A sample of labeled RNA containing C14 -. 9. how many Becquer els do you'think it has? 14. Ans. Why don't they use ap or asS or aH dating? " . He is enough of a researche r to know that every compound is not a glucose metabolite. Can you help him by pointing ou t his mistake? 18. Do you have a way out? 19. However. He labeled gl ucose with tritium homogeneously. A researcher is interested in studying glucose metabolism. the Geiger counter kept o telling you the truth? .545 17. Why do then researchers prefer using [3HI thymidine to a aP labeled nucleotide while assaying DNA synthe sis? 20. Both phosphorus and hydrogen . You have a 32p labeled compound and you have to measure it. he found that every single compound t hat he picked up has homogeneous distribution of the label. Still. you hage run out of some scintillation cocktail components. Was the researcher labeled a given compound with all. Unfortunatel y. Yo perspex shield covering the electroph out.are integral parts of nucleotides. Scintillation counting is based upon a phenomenon called phosphorescence . How is this different from fluorescence? 21. After suitable time he tried to detect the presence of tritium in various chemical compounds in the tissue. You put a oretic apparatus to bar radioactivity from coming utside the shield gives out noise. A researcher tells you that he has u take the substance from him and electrophoresce it. e. primer. The result was the development of PCR. When these obvious discoveries have been made at a later date many of them have turned out to be major advances. If the reaction can take place tn vitro. Inside the cell. template. and (fl0 addition of nucleotides on the primer in a sequence complementary to the template. ea ch recognizing one of the strands at sites bordering the sequence to be amplified a re mixed and the tube is cooled to 40°-60°C.. The tube also contains all four nucleoside triphosphates and a su itable . (//) provision of primer. the result of this cycle of reactions will be two double stranded molecules of DNA (see Figure 14. can one not select the region of the DNA to be replicated by synthesiz ing primer of a particular sequence? It is a pity that this question was not asked until 1985 wh en Kary Mullls of Cetus Corporation. dNTPs and purified DNA polymera se are provided? That it can was demonstrated by Arthur Kornberg in the sixties. Within the test tube isolated doubl e stranded DNA is denatured by heating at 90°-98°C to separate the two strands. asked it. the replication proceeds wit h (0 unwinding of the two strands. The primers anneal to complementary sequences (Figure 14. Obviously.2). i.1). within the nucleus. the reaction being carri ed out by DNA polymerase. Can this reaction not be carried ou t in vftro if all the requirements. DNA polymerase carries out the following re action (dNMP)n + dNTP (dNMP)n +I + PPi Thus. Polymerase chain reaction can be defined as recurring replication (amplification ) of a particular region of the genome preselected with the help of primers flanking the region of the genome to be amplified (Figure 14. USA. starting with a double stranded DNA molecule.2). Two primers. One such example is that of polymerase chain reaction (PCR ).14 CERTAIN PHYSICOCHEMICAL TECH NIQUES USEFUL IN BIOCHEMISTRY POLYMERASE CHAIN REACTION History of science is replete with examples of obvious and simple discoveries be ing missed. Each cycle of synthesis generates DNA products. and synthesis of DNA."t arget" DNA sequence. annealing. synthesis Of the complementary strand wi ll take place starting from each primer. If now DNA polymerase is added. ff repeated again and again leads to amplification of sdec ted regions of DNA. . or rather cycle. of denaturation. and these products become substrates of subsequent cycles of synth esis.buffer. which are copies of a. This process. mer attachment 5A.547 Parent double stranded DNA A G G TO A A T. G G T C A A'T A G A A C C3. Septed 3' G G 5' DNA sds . A G A AC C 5' " 3' T C C A G T T A T C T T G G Prt. the concentration of the "target" DNA doubles every cycle (Figure 14. I. . What this means is that after about 20 cy cles. when the mixture is heated to about 94°C. Nat urally taq polymerase is resistant to high temperatures. Due to this considerable amplification. As should be obvious. So far we have discussed all procedures with respect to DNA polymerase. Simplified diagrammatic representation of DNA replication. taq poiymer ase is used instead of DNA polymerase. which thrives in hot springs. the enzyme is destroyedthrough denaturation thus necessitating addition of a fresh aliquot at each synthesis step. this is why the method is known as a chain reaction. the concentration of the target DNA will be about a million times more (22°) than the concentration of nonamplified DNA. and Just one time addition of the enzyme is enou gh for any number of cycles. However. therefore. In PCR. 5' sded T C C A G T T A T C T T G G dauter A G G T C A A T A G A A C C DNA 3' molecules Fig. to be analysed easily. 14. Thus.A G G T pmem 5' atched plee A G G T C A AT A G A A C C 5' 3' o double 3'.2) leading to e xponential accumulation of the reaction products. it be comes possible to detect and to analyse small quantities of nucleic acids even when th e starting material consists of a large excess of sequences irrelevant to the investigator. the starting material can be as little as a few nanograms. This problem was solved with the discovery of Taq polymerase. th e DNA polymerase of the bacterium Thermus aquaticus. at-each denaturation step. of first cycle {-) .--) I Heat denaturation . l Renaturation (Annealing of primers) Primers (A & B} annealed Extend primers with Taq polymerase Completion.{+) 1 copy Separated strands 2 copies Separated strands 4 copies Biophysical Chemistry (. {iii) Taq DN A polymerase.(+) cycle {-) Figure 14.. Note that the number of copies increases exponentially every cycle. After the pr&ners have been extended by Taq polymerase. Temperature is lowered to al low the primers to anneal to their complementary sequences.Heat denaturation I Renaturation with primers I Extend primers with Taq polymerase Completion (+) of (-): second . . Essential components of polymerase chain reaction are (i) template DNA. DNA synthesis is initiated from two slngle stranded primers (A & B i n the flgure} flanking either side of target DNA and oriented in opposite directions. (ii} oligo-nucleotide primers. and (Iv) dNTPs.2 Basic PCR reaction which amplifies a target DNA sequence. Extension can now again be carried out. the DNA strands are heat denatured. and synthesis is done u nder two heads-the temperature at which each step is to be carried out. After this mixture is prepared. fie. After placi ng the tubes in the wells. a few drops of mineral oil are put in these wells so that the tubes are placed vir tually in an oil bath. this volume is not cr itical) of mineral oil is layered on top to avoid splashing of the mixture at high temperat ures. The temperatt!re at this . This is supposed to be good for avoiding contamination. About 4-5 units ofTaq polymerase. the Instrument and Precautions In a small eppendorf tube the following mixture (total volume mostly 50 gl or 10 0 gl) is prepared. and the time that each step is to be allotted. which accommodate the small eppendorf tubes snugly. reannealing. The DNA is added later by allowing the tip of the micropipette to go below the coil. extension or synthesis is always carried out at 72°C for about 2 minutes. which maintains a uniform temperature throughout the tube. Many investigators prepare a mixture of everything except the DNA and layer the oil.3).Lid Certain Physicochernical Techniques Useful in Biochemistry 549 Practical Procedure. It is th e reannealing step where the temperature usually varies. Programm ing of each step in the cycle. denaturation. The instrument has several wells (the number varies with instruments. Before putting the tubes in these wells. While denaturation is usually carried out at 94°C for 1-2 minutes. the lid is closed and the instrument is programmed. 10 x Taq buffer excess dNTPS excess 5' and 3' primers water to dilute the Taq buffer to Ix 1 gg DNA containing the region to be. amplified. The tubes are now placed in the instrument. a small aliquot (50-60 ml. which is known as the DNA Thermal Cycler (Figure 14.. the original instrument marketed by Cetus Corporation has 48 wells) . (Sample holder) Key board .step varies depending upon the A-T and G-C content of the primer. whic h is I°C below the melting temperature of the primer. The instrument also has the ability to store the tubes indefinitely at 0°-4°C after the programmed number of cycles are over and till the investigator is ready to take the tubes out. Usually 30-45 cycles are enough to achieve a satisfactory amplification. One cycle consisting of all the abov e steps can be completed in about 4-5 minutes. is selected for reannealing. the instrument will carry ou t the individual cycles automatically without any further act on the part of the worke r. a temperature. Once programmed. The time allotted to this step is usually 2 minutes. The hybrid DN A then needs to be introduced into the host organism where it will replicate. the investiga tor Joins a fragment of DNA to a vector DNA which replicates and produces multiple copies (s ee Figure 14. It can be easily understood that the contamination of the reaction m ixture with the smallest possible quantity of unwanted DNA can give an absolutely erroneous ampl ification. Table 14. The chances of contamination emerge out of the ability of the PCR to tremendousl y amplify the target DNA. Joining of the fragment of DNA to the vector DNA is necessitated by the f act that the desired fragment cannot replicate itself while the vector can.4 makes it clear that there are many steps.gene clo ning as production of a protein in large quantities for human use of course will not be replaced by PCR). after amplification. The preparation or" reaction mixture for PeR is best done behind a Perspex shiel d. It is good to confine the DNA thermal cycler into a secluded space and It is better If it is kept in a chamber having the facility of UV radiation so as to destroy DNA that may accJde ntally fall out. I PCR and Gene cloning: A Comparison . The final result of both PCR and gene cloning can be said to be a selectiv e amplification of specific sequences.1. the possibility of error in PCR is much less than in conventional gene cloning experiment.4). Moreover. Some major points of comparis on between PCR and gene cloning are listed in Table 14. if appropriate precautions are not taken . On the other hand. The precautions to be taken revolve around one central aspect: to avoid contamin ation. It goes without saying that the micropipettes used should be cleaned beforehand and afte r the preparation of the reaction mixture. Compr/son of PCR With Gene Cloning In many respects PCR has scored over gene cloning and it might be that PCR will ulUmately replace gene cloning to a large extent (such applications of . To achieve this objective via gene cloning.550. Figure 14 . The starting material that is required for gene cloning is afleast a thousand fo ld higher than required for a PCR experiment. both /n v/tro and /n v/vo in a gene cloning experiment. there isa danger of contaminating other samples in the laboratory with the PCR amplified s equence. /'-.PCR Clonin Ultimate result Selecting the target sequence to be amplified Concentration of starting material Requirement of biological reagents Manipulation Automation User bb/.f Labour intensive Cost Time required for a typical experiment Selective amplification of target sequences First step Last step Nanogram Microgram DNA Polymerase Restriction enzymes. ligase.. . vector bacteria In vitro " In vitro and in vivo Total None No Yes Less More .. 3-5 Hours 3-5 days . 551 Transform Certain Physicochemical Techniques Useful in Biochemistry Foreign DNA {Restriction site Digest with restriction enzyme shown by the bar) O O Vectorandforeign DNA anneal at the sticky ends Recombinant vectors carrying foreign DNA {Foreign DNA Indicated by wavy llnes) Figure 14. Bacteria carrying the desired recombinant plasmld are no w selected ago:inst 5'G A A T T C3' 3C T T AA GS' .4 Basic cloning procedure. Note that the vector (plasmld) has only one restrcthm site while the foreign DNA has several Restriction digestion is sequence specJlc and the cut ends are complementary (Eco RI cleavage site is given below) and upo n restriction ends of foreign and vector DNA can anneal through hydrogen bonding. The entire s et of recombinant plasmlds carrying different segments of foreign DNA are now introduced into the bacterial cells through transformation. Foreign DNA and vector DNA are cut with the same restriction enzyme (let us take Eco RI as representative example of restriction enzymes). There can be var ious variations to this technique all serving different purposes. It may be said with out the fear of exoneration that PCR has many utilities which are limited only by the investigat or's imagination. .Variation of the Basic PCR Technique In the above pages we have Only outlined the basic PCR process. A few of the variations of the basic PCR technique are described below. the DNA fragment of interest will be . there are times when the investigator has little idea about the sequence of the gene that he is interested in.5A). If PCR is now carried out with thes e primers. a knowledg e of the sequence of the gene to be amplified was essential in order to amplify it. H owever.5 B. Inverse PCR can also be carried out on linear DNA as shown in Fi gure 14. Region to be amplified cut with restriction enzym I Clorflng procedure Oved Insert e Region of unknown sequence to be amplified Region of known sequence .the vector DNA is known a nd hence one can synthesise primers specified for it. The inverse PCR technique is useful in such cases and will allow amplificati on of such a DNA fragment without using primers specific for it. In the basic technique described above.amplified and it will have sequ ence of the vectors at it ends (Figure 14.I Restrlct and llgate to circularize 0 552 Biophysical Chemistry (0 The inverse PCR technhlue. The In-st step in inverse PC R is to clone the DNA fragment in a circular vector DNA. The sequence of. fication product will possess known sequence at both ends. (B) In a linear DNA molecule. it is possible to amplify the sequences flanking t he known gene. Extension Amplified (B) product Figure 14. The amplt.5 Diagrammatic representation of inverse PCR (A) Foreign DNA of unknown sequence is cloned in a circular vector DNA. The DNA sequence flanking the known seq uence can now be amplified. . The primers which are complementanj to the ends of known sequence are annealed. Amplification product will possess vector sequence at both ends. Sequ ence of vector DNA is known and hence it is possible to synthesize primers complementary to those vect or regions which flank the cloned foreign DNA. if the sequence of the gene adjacent to the ge ne to be amplified is known (either partially or wholly}.Restriction site ) Primers annealed Extension Amplified (A} :product Primers annealed i. The DNA molecule is restriction digested and circularized by ligation. D. A single stretch of DNA is amplified in two different segments by using primer pairs A. Basic PCR reaction is similar to that'shown in. When denatured and reannealed.figure 14.553 1st cycle 2nd cycle (I) [1+2) Certain Physicochemical Techniques Useful in Biochemistry Denature aReanneal nd extend Figure 14. the amplified segments of the two PCR reactions form base pairing at the overlap region and can be extended in the next cycle to produce the whole original sequence. The base change is minor and does not alter the annealing of the primer with the template.6 Site directed point mutation by PCR. Internal ends of these two segments i overlap because the internal primers B and C are comple-mentary. B and C. Here.2. the primers contain the required base change ( indicates primer with one base change at the position of the cross bar). The base change is then faithfully copied into the new DNA strands in the subsequent. B D Figure 14.7 Site directed mutagenesis by overlap extension PCR. The primer at the overlap region (B and C} carry . cycles. alteration (s) is the base (s). (2) I Denature and reanneal I Extend Next cycle (Mutated product acts as the template) . Mutations can be induced at any region of the . altered internal primers for that region. e o R products e su at overlap e re. A meod. has been recently desired to overcome is on d is o as e o en . e meod consists of pg e pc gene as o sepate seents o sepate P CR reactions. It is ese t pers at a sequence vaflon(s) is Woduc depe n un e of mutation desed.tcggesi b//.7). s Hgaon step attaches a co--on nucleoflde sequence to e get sequence. ese ends e efore que to each ffent. howler. is is aceved by usg go e PCR reactions one per at e end of e gene d oer per at a suence mtem to e gene (re 14.on whe e mte p w sion d t erpp o memen to each oer. e per us cons a be wch ds not base p e template. e oer ends e deteed by vous chec cle mes or ch teaon (e.7].reacflon wch ows suence deteaon of R products ecfly.-spR eff respecve gent.554 Biophysical Chemistry |0 SIt directed m. (it0 $plg . e meod Is tHusated gure 14. For ple. .. e o PCR products e now ed d detured. A sple Hgaon step c be Woduced e blc R. e process Is sple d ows f deteaflon of e sequence. en re-eg is owed to e place. dldeo teaflon). Uge of s per is responsible for due t mu tation at a site cmted e posion ofe non-complemen base e per.. gure 14.8. () n Conveno R is not eately appRble to quen or footpg because e process reqes o deed ends. Each of ese ffen has one of e ends made on by usg eler a per or a rescon cut.produ. Such chec hoones eclt e acW of go e hoones whose genes were spced.6 shows flcy e prede for pg a sequence mumons oduced dung R itse.. e term pers used e o reacons e c omplen to ea oer. ers c be es such a way at stead of a pot mutation. ere e ser uses of chec gen es d e cec proteus. nscflon of cec r proteus have prodded pot oaon about e reons of genes pot for eir oncogec role. Usg a per to s coon suence. D e lese ch rcflon s mutation is copied into e dauter sd d er cles of R p e gene wch has e mumon. oer eple is at of coco n of a ceHc pepde hoone.e overlap enston me discussed m e prous pph ud to consct cec genes w 1 produce ceHc proteus. eension d s approach ows aesl s of hybd genes d mosc proteins. e site of e mtem pers be ved e gene depeng upon e site where e mutton is to be uc.7). PCR c be used to reduce mutations at a preselted site y ent of DNA d to p at mutated DNA. seons or deleons be oduc to e gene. e me is ed to we 1 effi mu site see mumons.g. is ted by e fact at see e pe used e te. e o overlappg sWds of e PCR pruc ne d eff overlappg ends now act as pers. e complete gene be sesd berg e mumon (e 14. s meod. howler. e ck is to use a per w ch is not peecfly complement. In e PCR at now be peHoed. nucleodes c be added upon d uque ends sesised just e conveno sequen cg or foot-p tecqCes. Usury a sequence or footpt ladder Is made up of related nuclelc acid frents. e mutations duced e gene so be at e ends of e gene. Using s meod It is possible to oduce mutton at at y site e gene of t erest (gure 14. 8 Gene splicing using overlap extension PCR. new applications are being added with an amazing speed. Applications There are numerous applications of PCR and since the technique is comparatively recent. We will not discuss the applications which are common and which can easily be imagined by the reader. specific. Primers A and D are dstnct but a part f prlmer C and B fs cmplementary t ea ch ther (heavy lfftes) The ampled products therefore overlap n thls regfon. These overlap re.al. B and C D. (1) Genetic diagnosis by PCR. In that very year. Kogan et. X and Y are two d'erent g enes and are to be spliced together for constructfon of a chimeric gene. forts act as prime rs in the next extension cycle and a chimeric gene formed. and highly sensitive diagnosis of sickle cell anaemia mutation can be achieved w ith fetal DNA samples using polymerase chain reaction. Upon denatumn and r enatumtWn mes X and Y form base pairs in this region. In 1987 Embury and associates reported that rapid. These genes are ampljcl usfng prlm er parrs A. Further ampl{flcation fs possible us ing primers A and D. Genetic diagnosis by PCR will soo . Instead some of the novel applications of PCR are discussed in this section.555 Certain Physlcochemlcal Techniques Useful in Biochemistry A Gene X C Gene Y Denaturatlon ednaturatio.n J Extension G no x Figure 14. describ ed a method for prenatal diagnosis of haemophilia A using PCR. Although.dfsm uslrtg/W. cells. techrticaliyspeaking.R. (C) less sample requirement. the worker expertise and sophisticated instrumentatio n required were not possible for small clinical labs. (B) lesser time requirement (see comparison of gene cloning with PCR discussed previously). PCR allows direct amplification of trace amounts of genetic material of infectious agents in blood. water.. These conditions were not required fo r PCR and this is where PCR scores over cloning and will allow genetic diagnosis to become commonl y available. PCR was achieve d by conventional gene cloning. most of what can be achieved by. Due to this abtlity PCR becomes especially valuable f or detecting . since purification is simply not needed. food. and (D) little or no loss of cellular DNA through prior purification. a nd other clinical and environmental samples. (i@ Study of/Ifetlous.n become a byivord in clinical studies because of (A) the easy nature of the experiment (PC R is totally automated). Som e examples of this use are cited bekw. Since hotspots of ras mutatio ns are known (12t. PCR is being tremendously used to gain Lnsights into the molecular basis of cancer. in the case of AIDS. The observation that activated (through single point mutation) ras genes are pre sent in a variety of human tumours has aroused tremendous scientific investigation of the role of these genes in tumorigenesis. 13t and 61at codons). and (B) m utation in ras genes occur at very early stages of tumorigenesis. chemical carci nogenesis studies on animals using PCR have been much more useful in demonstrating that ra s genes . PCR can be used to selectively amplify ras gene regi ons around these codons and the subsequent RFLP (restriction fragment length polymorphlsm) or sequencing allows demonstration of mutations fairly quickly. PCR can thus be of much use in pro viding a very early and almost foolproof diagnosis of the deadly disease that AIDS is. More importantly. PCR has also been succes sful in demonstrating that mutated ras genes are present in benign adenomas and malignan t carcinomas in the same frequency potentiating the belief that ras genes might be causative rather than secondary genes for carcinogenesis. Nevertheless these same persons may te st positive later on. [iiO in ¢¢m" research. Howe ver. However. PCR can detect the presence of even a small number of HIV and its genome even though no immune response is detectable. thyroid follicular carcinoma (50%}. causa tive of AIDS. colon adenoma or adenocarcinoma (50%). it is known that many in fected persons do not test positive for AIDS antibodies. and seminoma (40%).556 Biophysical Chemistry pathogens that are difficult or impossible to culture {examples . In order that-ras genes be demonstrated as causative for tumorigenesis and not merely of secondary involvement it is necessary to prove that (A) mutate d forms of these genes are consistently associated with a particular tumour type. Using PCR it has therefore bee n possible to demonstrate that mutated ras genes can be consistently associated with pancreati c carcinoma (90% pancreatic tumours contain activated ras).the HIV. Moreover. PCR is not yet of much diagnostic value for this human malady. the agent for lyme disease). Since clinical markers of predictive value are not available for cancers. When 2-day old rats are injected with nlt rosomethyl urea. ms mutations in the mammary tissue can be demonstrated as early as at the stage of 15-days. (iv) l'io u ofFeR. develop only after onset of puberty. ha/r found at the icene of or/me can be analyled by PCR amplttlcation and can provide a clue to th e ident/ty of the crlmmal when matched with the DNA of the suspects. PCR is also being used to screen mutations in tumour suppressor genes such as th e retinoblastoma and thep53 genes. Presence of viruses that are implicated as causative agents for human cancers ca n be demonstrated by the use of PCR. it may be said that once clinical markers of predictive value become kno wn. Gs can therefore be viewed as a pot ertial oncogene. the protein that transduces signals from [-adrenergic receptors to adenylate cyclase. however. blood. It was thought that in pituitary tumours the elevated levels of cAMP cause uncon trolled proliferation thereby contributing to malignant transformation. Using. PCR will provide an excellent early diagnostic warning so that the patient can be tr eated at a very early stage.are activated early in carcinogenesis. Samples contalnlngas less as I00 DNA molecules c an be screened for the presence of such viruses. PCR it wa s recently demonstrated that pituitary tumours contain mutated versions of the a-subu-nit o f Ga (mutations in the 201st and 227th codons). PCR has been used to iden t rrstng . DNA of trace materials such a semen. Earliest turnouts. Lastly. Just 6-10 cell cleavage.Physlcochemical Techniques Useful in Biochemistry 557 . The ability of the PCR to amplify ancient DNAs open up the possibility of stu dying molecular o|utlon by actually going back in time and dlrecfly approaching DNA sequences th at are cestral to their present day counterparts. A rough dating of the ancient sample m. Recently. Another area in which the utility of R is increasingly being realised is the matching of transplant donors and recipi ents. selection and metic drift can be studied with PCR even if small number of preserved samples ar e available. Evolution of population size. is widely b elieved be ancestor of the modem day elephant. the . What is more . using PCR and direct uencing. which is now extinct. The mammoth se quences roved to be closely related to the modem elephants thereby proving the link betw een these o. ivlfJ Other applfatlana. migration. (vO Archueology and anthropology using IR. The mammoth. This makes possible for us to relate many extinct les to other extinct or extant species.rsons and to settle paternity cases through DNA analysis. When they arc with the sequences of modem day Egyptians. Similar studies involving amplification direct sequencing studies are in progress with Egyptian mummies. (v) Study of evolution using PCR. PCR enables the researchers to determine the sex of an tn fertilized embryo at a very early stage of. mitichondrial sequences from 7000 year old brain excavated in Florida w ere mpared to mitochondrial sequences of more than 100 present day native Americans. Determination the extent of change in the sequences of the introns of the ancient DNA as compa red to modem DNA of that specie might give a rough idea about the age of the sample. the study will yield important about the population structure and geneological history of the Egyptians.. It found that this ancient DNA sequence does not match any of the three mitochondri al ' found to date in America. PCR amplification allowed DNA from Siberi an mnmoth (these mammoths have been well preserved since they have been buried in t he ice ra long time) to be compared with the DNA of modem day elephants. Further studies are on to find out the population and geneological history of Amerindians.ay be possible using PCR and direct It is known that sequence changes in the exons of same genes of the same are much rarer over a period of time as compared to the introns. The sensitivity of PCR allows one to amplify a gene from a single isolated cell. (D) Easy and extremely quick operation. . These genes can then be cloned bacteria that will then mass-produce the particular antibody. can only/mane the range of appl/caUons that would be ava/lable a decade later. The PCR technology thus hybridoma technique redundant for the production of monoclonal antibodies. (C) No skill required on the part of the worker. even a single cell may be sufficient sometimes. This can be well employed to pick out a single antibody-producing cell and to amplify the genes encoding the antibody.does not interfere with the life of the embryo at all. The many advantages of PCR technique are summarized below. (B) The ability to amplify this DNA seglnent from an extremely small quantit y of sample.Ability of selectively amplifying a small segment of DqA from a sample cont aining numerous unrelated sequences. This approach will be ver y for genetic counselling and avoiding birth of a girl in those cases where the pa rents at a risk of X-linked disorders in their offsprings. These advantages of PCR have made it one o.f the most prime developments of our The appl/eations of PCR are numerous even at this nascent ste oi the technology. (A) . In Chapter 13 we have discussed radioimmunoassay. The second immunoreagent is li nked to an enzyme in a way that there is no loss either to immunoreactivity or to the enzym e activity. The reagents for ELISA on the hand are long lived. a chromogenic substrate of the enzyme is supp lied. After incubation and subsequent washing. Dueto the problems the radiochemicals might pose to the hea lth. The basic principle of E LISA is shown diagrammatically in Figure 14. colour will develop because of the presence d the linked enzyme. The principle of ELISA is as follows: one of the immunoreagents is immobilized through adsorption On the solid phase support (usually polyvinyl chloride or pol ystyrene) n such a way that there is no loss to its activity. a technique used to detect bioactive antigens and even antibodies. If the two immunoreagents have bound to each other. 0 Subtrate (color less) " Ev.558 Biophysical Chern/stry. ELISA is fast replacing radioimmunoassay s because certain inherent advantages.9 Prlnctple of Enzyme Linked Immunosorbent Assay. .9. ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) ELISA is today playing an increasingly important role in diagnostic and research laboratories the world over. Fir@t of all.zyme A Product (colored) Conjugate Adsorbed Reacting " antigen antibody Solid phase Fjure 14. ELISA does away with the need of which are difficult to store because of their short shelf life. ff not there will not be any colour. .. Moreover. ELISA lends itself sophisticated automation and is quite adaptable to simple tests. The plates are subsequently washed to remove unadsorbed antigen and then dried. Discussed below are a few assays which are routinely used. If the antibody specific to the adsorbed antigen is present it reac ts . with a variety of detection systems such as visual comparison. Salient points of the procedure for indirect method are briefed below. for examp le enzymelabeled antihuman globulin. photometry. and luminometry. for example enzyme-linked antihuman IE. The method has become popular because it requires only a single conjugate.SA can be. Different immunoglobulin classes can be detected dif ferentially by using class specific conjugates. ELI. These plates can be stored. (ll) Diluted serum (or any other test sample) is then filled in these wells a nd allow to incubate. Lastly. as administrative inconveniences are circumvented.is limited by legislation. fluor ometr. (i) A suitable antigen is allowed to adsorb passively onto the wells of plas tic microplates. Assays for Antibody: Indirect Method This method is largely used to measure antibodies in almost all human infections . There is no such health risk with ELISA reagents and. The test sample is then incubated with t his phase and subsequently the enzyme linked antigen conjugate is reacted. Washing is now carried out to remove all unreacted serum components. It is summ arized (i) A specific antibody is immobilized on the solid phase by adsorption. A summary of the above steps is given below: Solid phase-antigen test sample antibody anti-immunoglobulin enzyme conjugate + substrate colour An alternative to the indirect method is the so-called Sandwich methocL In this method the antigen is adsorbed to the solid phase. (///} Enzyme-llnked anti-human immunoglobulin conjugate (or any other class sp ecific conjugate) is now added to the wells and allowed to incubate. A chromogenic enzyme substrate is now added to the wells in a solution. The reaction is stopped after a particu lar time and the colour intensity is measured photometrically. Finally.Techniques Useful in Biochemistry 559 and becomes immobile. (ii) The test sample containing the antigen is now incubated with the antibod y immobilized on the solid phase. Unreacted antigen is washed away subsequently. (iii) Enzyme-linked antibody specific for the antigen is now incubated. for Antigen: Sandwich Method The sandwich method is the most largely used method for this purpose. (iv) A chromogenic substrate is now added and the colour rnasured by photomet ry. . The rate of degradation of the substrate (increase in colour intensity) is proportional to t he concentration the antibody in the serum. The unreacted conjugate is washed away. The un reacted antibody conjugate is then washed away. the is added to develop colour. This conjugate now binds to the antibody captured on the solid phase in step (ii). is influenced by the material used for the solid pha se.biotin-avidin system. the and the concentration of the immunoreagent. Polystyrene is therefore the material of choice when non-specific with serum components is to be minimized. by the and finally by the time of incubation.reacted. The above sandwich method is followed as it is up to the incubation 'test sample reagent with the solid phase [step (ii)]. Solid phase-antibody test sample antigen antibody-enzyme conjugate + substrate .The colour intensity is proportional to the concentration of the antigen. So. Subsequently a biotin-lab eled antibody . Of these polystyrene binds much less protein as to the latter. lid phase-antibody test sample antigen conjugate avidin-enzyme conjugate + substrate colour antibody-biotin Practiczl Notes Coating of the solid phase. Finally an avidin-linked enzyme solution is incubated. by the nature of the diluent. Two types of solid phases are in general polystyrene and polyvlnyl chloride. colour A variation to this method to increase its sensitivity has been made through the use . On the other hand when a high . periodate. this . the colour changes to orange-brown. However. and avidin can be linked to the enzyme throu gh glutaraldehyde. glucose oxidase. How ever. The test sample concentration should be adjusted so that the measurement is opti mum. The coated phase can be stored in a de ssicator in a refrigerator for periods exceeding five years with little loss to the reactivity . penicilinase. polyvinyl chloride is the best solid phase. Another problem which should be avoided arises due to non-specific reactivity of the sample comp onents which get adsorbed directly to the plastic surface. Usually the test samples have to be diluted to reach the optimum concentration. This enzyme is cheap and can be attached to the immunoreagent by a v ariety of methods. 8" galactosidase has become very popular now since it can be used with fluoro substrates. urease etc. When the reaction is stopped by addition of acid. phenylenediamine (OPD) which produces a strong yellow product. with polyvinyl chloride eve n one hour incubation at room temperature is enough. To reduce this problem a wetting a gent such as Tween 20 is usually included in the incubation medium. The most often used enzyme to be conjugated with the immunoreagent is peroxidase. Usually overnight incubatio n between 40 to 8°C is sufficient to coat the solid phase. maleimide. Mos t often BSA used as the blocking agent. Moreover many chromagenic substrates for it are also available. The purit y of the immunoreagent is a must since impurities. If biotin avidin system is followed. The diluent used for proteins is usually carbonate or phosphate buffer used either at a neutral or alkaline pH.560 Biophysical Chemistry coating level is required. biotin can be linked to th e antibody incubation with dimethyl formamide. The substrate is such that it is colourless before reaction and after degradatio n produces colour. Conjugates are usually prepared with glutaraldehyde as the linking agent. The conce ntration used normally for proteins varies between 1-10tg/ml. Along with the wetting a blocking agent has to be used to further minimize non-specific reactivity. N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) have also used as linkers. wil l compete for available space in the solid phase surface. if present in substantial amounts. The substrate used most often with horse radish peroxidase is o rtho. enzymes are alkaline phosphatase. Alternativ e substrates for peroxidase are 5-aminosalicylic acid. 5. particles is known as cytometnj. The absorbance of the solution can then be easily measure d using an ELISA reader. 3. in general. When such measurements are performed while the cells other biological material pass in a single file through the measuring apparatus the process is known as flow cytometry. Some fluorogenic substrates have become popular of late. These are methyl galactoside for the enzyme -galactosidase and methyl umbilliferyl phosphate for the enzyme alkaline phosphatase. 5 -tetra methylbenzidine (TMB ) and 2. Normal ly the incubation time is about 30 minutes following which the reaction is terminated b y either alkali or acid. 3. The intense interest centred around flow cytometry is mainly due to the speed which analyses are performed (even up to 3 x 105 cells per minute) and due to th e .2azinodl (3-ethyl-benzithiazoline sulfonic acid-6) diammonium salt (ABTS).substrate is light sensitive and there are doubts about its safeness. FLOW CYTOMETRY What is Flow Cytometry? Measurement of physical and/or chemical characteristics of cells or. An additional f unction that flow cytometers can perform apart from cellular analysis is their physical sorting.2). T his sorting takes Just few minutes and gives purity of any cellular subtype in excess of 95%. It is very im portant to note that all the measurements are performed on a cell by cell basis. Pylieal Paxamete Chemical Paxamete Cell size Cell shape Cytoplasmic granularity Cytoskeletal organization Redox state Membrane integrity Endocytosis Surface charge Membrane fluidity Structuredness of cytoplasmic matrix Membrane potential of biological membranes Animal and plant pigment content Total protein Basic protein Sulphydryl groups DNA content DNA base ratio DNA synthesis RNA content Antigens surface sugars .561 of simultaneous analysis of multiple parameters (see Table (14. Typical measurements inclOde electronic cell vo lume. and fluorescence polarization. light scatter due to extrinsic or intrinsic cellular features. fluorescence of endogenots cell constituents or from stains bound to cell consti tuents. functional. cytological relationship to be established between different cell ty pes.Enzyme activity Membrane permeability Intracellular receptors Surface receptors Membrande bound [Ca Cytosolic [Ca2+l Intracellular pH The flow cytometer analysis and sorting instruments combine electrical and optic al sensing techniques thereby permitting several measurements to be performed simultaneously on the same cell. absorption or los s of extinction of light due to cellular components. saline.impinging upon them. Cells incuba ted with fluorescent or absorption dyes are suspended in. The basic process of flow cytometry is illustrated in Figure 14.10. the simulteaneous measurements allow a biochemical. Sign als from each of the sensors are processed on a ceil by cell basis and the resulting data are displayed as frequency distribution histograms. Additionally electronic sensors detect the particle volume and other related parameters. If a hetero geneous cell population is being assayed. . they either scatter. This sc attered. or fluoresce the light. absorb. A s the cells pass. physiological. transmitted or fluoresced light is then measured by appropriate optical sensors. Thi s suspension is allowed to pass through the flow chamber at a rate of about 1000 cells/see.. 10}. all of them are composed of the same basic units. The measurement region means the site where the cell stream intersects an excita tion beam of light (Figure 14. signals pr ocessing electronics. This region i s usually located inside the flow chamber but in certain instruments the measurement regio n is placed outside. fluid transport system. which increases the flow velocity . Instrumentation Although a variety of commercial flow cytometers are available. stability and spectral purity. Abo ut 2% of such droplets contain one cell each. Measurements on cells are made here. The droplets. flow chamber. The sample along wi th the sheath fluid now enters a constriction region. optical and electronic sensors. The cells from here are pressurise d for delivery by tubing to the flow chamber. If the amplitude of the signals fall within a preset range a droplet cha rging device is activated. v/z.flow cytometer After measurement the stream which exits from the flow chamber is disturbed by a peizoelectric transducer to give uniform droplets at the rate of 40.562 Figure 14. In the flow chamber the sample stream is injected into the centre of a ceil-free stream of sheath fluid (Figure 14. In order that the cells pass in a single file. which are so charged. and data display.10}. collection optics.10 A highly schematic representation of. storage and analysis system. excitation source and optics. The samples are placed in a sample chamber which is fitted with a Continuous sti rring device to mix the heterogeneous cell population and with a filter to remove cell clumps from flowing into the fluid transport system. it is necessary to adjust the sample conce ntration and the sample stream diameter. In additio n it is easy to . Lasers hav e the advantage of extremely higll radiance. are deflected by a static electri c field and are collected in a container. For optimum measurements it is necessary that a laminar flow be maintained. The source of light in most modern instrument is the argon-ion laser. Other droplets not containing the desired cells are no t charged and these are therefore not deflected.000/sec. measurement region. One can achieve a cell separation rate of 100 0 cells/sec and purity of such cell preparations is routinely about 95%. Processed signals from cell sensors now activate the cell-sorting device. .focus them to areas equal to the cell dimension A beam stop is placed to stop th e laser after it has irradiated the cells. Appropriately positioned detectors measure the fluores cence and light scatter due to the cells. (tO the ori entation of the dell in flow. width. The higher angle light scattering assists in the identification of specific cell sub-popula tions.Mithramycin. and (iv) the internal structure of the cell. stores. the peak gi ves us the about the maximum fluorescent intensity of the cell. Usual placement for fluorescence detectors is at 900 to the inciden t laser beam. For every pulse. the width tells us about th e width of the fluorescing part of the cell and the area of the pulse informs us about the total fluorescence of the ell. This electrical pulse is amplified and converted to a digital value before being sent to a digital computer which analyses. It might be stated here that fluorescent measurements are the most often used . A se ries of detectors and filters are positioned appropriately to measure fluorescence at a number of wavelengths. (/to the refractive indices of the cytoplasm and the nucleus. or displays the signal. Analysis of low angle forward scatter therefore reveals much inform ation about the above cellular parameters. its peak amplit ude. oxazlne I. acridine orange. pyronine Y. Low angle forward light scatter is dependent upon (t) the cell size and shape. Each detectr in the flow cytometer gives a signal in the form of an electrical p ulse that is proportional to either the concentration of a cellular substance or to a cellula r feature. chromomycln. Many instruments also use two or more sequential lasers to excite fl uorescence in cells at multiple wavelengths. If fluorescence measurements are being performed. olivomycin. orthogonal (90°) light scatter gives impor tant information about the granularity and fine structure of the cell. Fluorescein isothiocyanate [FITC] Certain Phtjsicochemical Techniques Useful in Biochemistry 563 Most of the modem flow instruments are capable of multiparameter detection. Ligh t scattered at different angles reveal different information about various cellular characte ristics. propldlum Thlofalvln T. The presence of multiple detectors allows use of several fluorochromes to detect multiple cellular parameters. eth/dlum. The cells passing through the flow chamber also scatter the incident light. and area are recorded. In addition. Thus a flow instrument con tains a series of detectors to measure the light scattered at different angles. 2'-dlsulfonlc acid (SITS) dansyl chloride indo. Fluorescein based substrates . A list of fluorochromes used to assay DNA. fluoresceln d/acetate Carboxyfluorescein dlacetate. 4-methylumbelliferone Coumarin and naphthol based substrate. SITS.6-dlamino-2-phenylindole RNA content Total Protein Cytoplasmic [Caz'] Intracellular pH Enzyme activity Surface structures Membrane potential Membrane integrity 4-acetamldo-4'-isothiocyanatostilbene 2. RNA.3.4-dlacetoxy-2-3-dicyanobenzene.measurements in flow cytometry. proteins. Table 14. dansyl chloride Oxycyanines Prlmulln.1 1.3 Some Fluorescent Probes Used in Flow Cytometry of Important Cellular Parameters Parameter Fluorescent probes used IJV excitation Visible excitation DNA content 4'. or various cellular features is provided in Table 14. Rhodamlne 123 Propldium. Oxacarbocyanines. fluorescein diacetate . Phycocyanine. indocarbocyanines. phycoerythrin.FITC. ailophycocyanlne.texas red. (4) T of anul d plalt n. For example Chinese hamster embo celia were treat th the fluorochrome propidium iodide. d granuloces and to so them out. Tests for ulote function such as NBT reduction. (J $ on hmom. Ofily a few representaUve appcations have been cited above. Moreover. Flow omet mes it easy to quanfl cell-to-cell communication. Iph omas etc. monoes. (3) uocte characterization. (5) an of ¢ell-tell ¢ommunivan. e sfer of e o dyes beeen donor d recipient cells c be studied easily using two colour fluorescence flow omet. major well documented appcaflons have been cury det . Such studies therefore achieve great siificance in studying malignant celia that often demonstrate altered kote. genetic counselling can also benefit from flow ome as th e altered kope c be studied we easily and quicy. and superode is generated duflng phagoosis by neutrophils. dichlorofluorescein oxidation. A deled discussion of ese appcaflona is imp0ible here. Using flow ome it has been demonsated at oplasc Ca* concentration increases. e chromosomes were then alysed by cig at 4 nm d studg e fluorescence at wavelena above 560 . It so becomes possible to so ce popaflons erent ce de phases d to s ubject each popaflon to bioche ysis. Cells are scrape-loaded th Lucifer yellow th or thout rhodamine labelled d. Platelet activation c also be measured easily by measug crse oplasc Caa+ concentraon. As an example work of va may be cited. membrane potential. By eension it c be sd at tered kaotype would gi ve an altered fluorescent pattern. However. Platelet abfli c be assayed easily by eler a dye exclusion test or by measug e membre poten usg a cboe dye. In addlUon It may be . It is possible to detee kaote of a cell usg flow cytomet. Hi speed comosome soers are now use to facilitate e task of mapping e hum genome. Acdine orge Is so ued to chactee leukoes from ents vaous nds ofneoplasma such as leukemlas. 11 chromosomes gave a derent pe. [} .564 Bphysal ChemLstry APllieltions of Flow ome Flow ome h numerous appcaons and as case of PeR it mit be sd at e appcations of flow ome e ited oy by our o imaaflon. d phagoic assays are I possible usg flow ome. it comes possible to dee ces' posion e cell cle. In addlon flow come Is so utilized for derenfl leoe countg where it ves we precise vues. measurement of deulation. membrane potentlal becomes more positive. sus measmen ofDN A. ce s d prote. o colour fluorescence characterization of acridine orange treated leukocytes makes it possible to dlstinghlsh between lphoes. parasitolo. tissue ting d lphoe applications. cancer blolo d carcinogenesis.sd that flow comet finds numerous applications in e fields of ce differentiation . bone mow analysis. sperm analysis. immunolo. d T-ce subset anysis. . food science. phaacolo and tocolo. Certain Physlcochemlcal Techniques Useful in Biochemistry 565 Flow cytometry is increasingly being used for sorting different cellular populat ions.N. H. 5. Thus. Gene. and Wood. Jr. Horton. J. and White. B. Current Communications in Molecular Biology. (Erlich . M. Principles and Applications for DNA Application..B. (I 990}: The Unusa/Or/gin of Polymerase Chain Reaction. and Koshland. A Guide to Methods and Appllcatlons (Innis. D.J. and Hunt. S. Nature. D.R. Science. H. eds. 9..J. et al. and Hunt H. (1989}: The Mo/ecu/e of The Year. 8.. et aL. different leukocytes.. et al. 246:780-786. ed. 335:414-417. R. U.D..B... Gibbs. Jr. Gelfand . macrophages.}. 1989. Eng/neer/ng Hybrid Genes Wit hout the Use of RestrlctWn Enzymes: Gene Splicing by Over/ap Extension. (1989): Site Directed Mutgenesls by Overl ap Extension Using the Po/ymerase Chain Reaction. 1989. 6. New York. Sci entific American. and many other types of cells are routinely s orted using flow cytometry. (Erlich.A. Science.E. H. 246:1543-1546. Academic Press. (1988).L. 77:51-59. Polymerase Chain Reaction. PCR Technology.. R. San Diego. 4. Gene. Guyer.M. Mullis.H. 42-49. 7. Ho. 3. Cold Spring Harbor Laboratory Press. Suggestions For Further Reading I. K.). P. Sninsky.. and Kazazlan. 77:61-68.D. (1989). H. . PCR Protocols.) Stockton Press. (1989}. eds. Li. H. 2. Gyllensten. In vlvo Footprlntlng of Muscle Specifi c Enhancer by Ligation Med/ated PCR. R. Aprll. T. Muller. 1989. Amplification and Analysis of D NA Sequences in Single Sperm and D/p/o/d Ce//s. Liss Inc.. Sci. 3" Ed. Frei dman. and J. Vikas Pu blishing House. and Pulmann. G:P.. New Delhi. 2"d Ed. Manual of ClinicaILaboratory Immunology. 14. (Noel R.) American Society for Microbiology. 109:129-135. Fahey.M. 13. Engvall. 12. Instrum... H. Weinstein eds. Diagnostic Flow Cytometry (John S. 1986. SteinKamp. EIISA III QuantltalWn of Specific Antibodies b y Enzyme Linked Irnmunoglobulin in Antigen Coated Tubes. 15. (1984): Flow Cytometry.Rose. I I. Rev. 55:1375-1399. J. Baltimore. P. 1972.. Alan R.10.99-109. B. Shapiro. eds. Talwar. Non-isotopic Immunoassays and Their Applications. 1983.. Coon and Ronald S.) Wi lliams and Wflkins. (1988): Practical leww Cytometry. New York.. H. A. pp. J..L. ImmunoL. 1991. . APPENDICES . 42 .1 28 (as NH3) 1.9 15.5 19 50 1.9 96 1.84 35.Ammonium hydroxide (stronger) Sodium hydroxide Isaturated] Sulphuric acid (concentrated) Nitric acid (concentrated) Hydrochloric acid (concentrated) Phosphoric acid (syrup) Acetic acid {glacial) APPENDIX A Specific Gravity. Normality and Per Cent Concentration of Common Acids and Alkalies as Purchased AcidAlkali Specific Gravity Normality Per Cent (room temperature) by Weight 0. 06 17.5 1.15.6 69.8 .6 99.19 11.71 45 85 1.7 36 1. 60 162 Table 2 Aconitic acid-sodium hydroxide 100 ml 0.00 65 1.5 M aconitic acid + X ml 0. 23oC.2 M KCI.40 266 2. diluted to 1000 ml. 1. Temperature 25°C. I0 528 1.70 130 I.30 336 2.00 670 1. diluted to 1000 ml.20 39 1.50 207 2..20 '425 1.2 M HCI + 250 ml 0.570 APPENDIX B Composition of Some Routinely Used Buffers Table 1 Hydrochloric acid-potassium chloride X ml 0.90 81 1.80 102 1. .10 51 1.2 M NaOH. 5 75 4.0 465 35 4.5 26O 5.7 485 3.3 565 3.7 105 4. diluted to 1000 ml. 23°C. pH X Y pH X Y 3.9 515 3.6 .9 340 5.1 180 4.1 M citric acid + Y ml 0.2.9 140 4.1 54O 3.5 595 " Table 3 Citric acid-sodium citrate X ml 0.1 380 2.3 415 2.1 M Na citrate.5 450 3.3 220 4.7 300 5. 0 330 170 5.6 137 363 4.4 280 220 6.0 95 405 .0 205 295 3.6 370 130 5.8 118 282 4.2 437 63 4.2 180 320 3.4 160 340 4.4 400 100 5.8 350 150 5.255 245 3.8 230 270 3.2 315 185 5. 23oc.0 267 4.2 M NaOH.8 75 5.2 303 4.2 .2 133 5. pH X p/-/ X 3.0 I00 5. diluted to 1000 ml.4 342 4.0 435 Acetic acld-sodium acetate X ml I M actlc acid + Y ml I M NaOH.05 1=0.1 =0.8 235 6. (I = ionic streng th) =0.6 200 5.6 375 4. 25oC.8 407 4.2 M succinic acid ÷ X nd 0.Table 4 571 Table 5 8uccinic acid-sodium hydroxide 0. diluted to 1000 ml.4 167 5. 2 65.6 650.0 4.2 200.8 428.0 389.0 559.0 423.0 283.0 .O -4.8 146.6 I I 0.0 553. 341.0 --3.pH Y= 50 ml Y= I00 ml Y= 200 ml 1 M NaOH I M NaOH 1 M NaOH X X X 3.0 4.0 828.0 760.0 288.0 5.0 289.8 87.0 215.7 173.0 73.0 .0 -4.0 5.4 145.0 4. 8 102.6 290 6.2 .8 53.2 157.5 I 18.0 5.4 270 5.6 56.2 M NaOH diluted to 1000 ml.0 222.2 255 5.4 54 7.3 -Table 6 Trls acid maleate-sodlum hydroxide M tris acid maleate + X ml 0.0 240 5. 23°C.0 112.5 6.0 256.6 77.5 7.4 185 8.0 345 6.0 235. 5.0 5.4 59.5 8.8 317.2 35 7.5 7.0 5.0 130 7.8 107.129. 8 212.4 405 6.8 225 8.5 .375 6.6 432.5 8. 8 6.2 8.2 64.58 6.4 28.8 7.5 M K/PO4 + Y ml 05 M NaHPO4.0 22.5 6. d/lUted to 1000 ml.4 22.6 11.3 6.8 31. I I= 0.8 7.0 74.Table 7 Table 8 Biophysical Chemistry Potassium d/hydrogen phosphate-d/sod/urn hydrogen phosphate Xni. 0. 25°C.2 .2 15.0 19.2 pH X Y X Y X Y 5.8 --6. {I = ionic strength) =0.6 42.05 I=0.4 25.4 53.4 15. 0 7.4 48.74 31.8 222 59.8 17.38 64.2 333 22.7.8 4.4 .36 31.6 60.8 142 19.0 7.4 55.8 8.5 62.4 159 13.80 32.0 53.6 26.3 30.6 6.0 75.4 98.5 121 26.4 303 324 265 44.2 39.4 57.8 l 1.4 10.8 7.6 41.0 2.2 34. 4 .44.1 M NaOH. diluted to 1000 ml.1 68 7.8 111 .9 46 7.2 6.1 M KHPO4 + X ml 0. 25°C.0 56 7.2 i02 65.2 119 29. pH X pH X 5.1 129 Potassium dihydrogen phosphate:sodlum hydroxide 500 ml 0. 89.176 74.9 127 12.8 36 7.1 6.6 133 .2 81 7.0 5.2 95.2 124 18.3 6. 4 116 7.7 193 7.3 97 7.0 6.7 6.5 139 7.6 164 7.8 6.5 6.9 259 -291 321 347 370 391 309 424 435 445 453 461 .6 6.9 6.6.8 224 8. 5 257.0 8.0 7.0 208.0 8.1 117.6 117.0 86.3 222.I I=0.8 235.0 .2 55.2 X = 5O rnl I M HCl X = I OO ml I M HC[ X = 2OO mI I M HCI Y Y Y 7.Table 9 'lble 10 I= 0.2 107.0 53.0 342.0 421.9 144.2 214.05 I=0.0 8.0 290.6 64.1 169.4 59.4 127.0 7.3 7.0 7.4 141.8 72.7 111. Diethylbarbitur/c acid-sodlum 5:5 .0 8. I /=0.0 -9. diluted to 1000 ml.6 194. (I = ionic strength) /= 0.0 524.0 367.5 M barbltone sodium + Z ml 0.0 --5:5' .270.2 627.2 X Y Z X Y Z X Y Z 648 I0 90 409 10 90 645 25 75 .0 550.05 I=0.5 M NaCI.0 -9.0 738.8 279.0 8. 25°C.0 414.diethyroarblturate (Barbitone-barbltone sodium} X ml 0.025 M barbitone ÷ Y nd 0.0 761. 514 50 50 648 I00 409 I00 -258 I00 -163 100 -639 I0 190 403 10 190 636 25 175 401 25 175 506 50 150 639 I00 I00 403 100 100 509 200 -321 200 -- -- .814 50 50 . 275 5 395 348 I0 390 219 10 390 346 25 375 218 25 375 275 50 350 348 I00 300 438 200 200 277 200 200 . 'L 0"6 O'LIt.'9I I 6"9g 9"9 6"9I 'OI I 9"gg 6"II -- .--O'g19 I'Ol -O'gL O'OI g'Ol -O'gl O'ILg 0"01 O'IL9 O'09g O" 161 9"6 0"661 O'I9g 0"6gI 9"6 0"69g O'IOg 0"90I I'6 0"6I 0"9I '9 '6 O'gLE O" IF I. 0"9I [ "I9 9"9 0"0 I. -'9 I'6 0"6 6"8 9"9 '9 '9 .'8 0"9 X Hd x Hd . diluted to 1000 ml. I 116.0 467.1 I = 0.0 9.575 Appendices Table 13 Glycine-sodium glycinate X ml 2 M glycine + Y ml 1 M NaOH.6 58.05 I = 0.0 .0 313.0 437.4 155. 25oc.1 216.2 108.1 234.0 9.2 Y= 50 ml Y= I00 ml Y= 200 ml 1 M NaOH 1 M NaOH 1 M NaOH pH X X X 8.0 9.8 234.4 77.0 312.0 637.0 9.0 157. (I = ionic strength) I = 0. 3 153.6 28.7 185.9.2 X Y X Y X y 9.5 134. (I = ionic strength) I= 0.8 26.5 .8 45.4 30.0 10.8 91. 25oc.3 121. diluted to 1000 ml.0 10.7 53.8 3.0 56.4 113.0 I0.0 38.2 33.I I= 0.8 108.1 60.0 Table 14 Sodium bicarbonate-sodium carbonate X ml 1 M NaHCOs + Y ml 1 M NaCO8. I 10.0 10.0 39.2 66.05 I= 0.1 76.2 35.41 9. 9 24.6 25.3 6.4 45.8 18.3 ¢3..2 34.8 14.4 10.8 56. 76.9 10..8 2.4 6.74 67.0 .6 3.8 7.6 24..86 15.4.6 11. 12.0 13.29 14.2 10.5 10.0 " 27.7 II.6 10.58 13.4" 30.83 9.2 29.0 10.2 9.5 .3 18.01 15.6 8.3 21.55 9.44 9. I 17. 5 42.8 127.1 12.7.4 104.0 17.9 19.06 30.4 31.3 29.9 52.0 24.8 407 31.4 7.9 81.92 64.9 3.2 56.9 .41 63.5 39.3 147.3 60.5 46.80 32.7 1.5 60.3 62. 0 3.90 50.576 Bloptu3siccd Chemlstr Table Un/versa/buffer m/xture 1000 ml of a mixture of diethylbarbituric acid.0 8..68 35.14 65. pH X pH X 2. and boric a cid.0 8.0 5.2 M NaOli.92 15.36 I0.27 30.71 80.15 75.90 5.0 5. KIIPO.0 7.40 20.82 25.50 85.0 2. 25°C.0 3.0 4.71 60.0 9.30 55.0 4.0 10. citric acid. 0.0 9.02857 I/with respect to each of the above compound + X ml 0.0 .0 7.63 70.42 0.0 6. 10 40.25 90.0 11.51 45.0 .8.58 95.79 I00.0 11.0 6.0 11. "Img 1 g Ing Ipg APPENDIX C I Unit Name metre second kelvin mole ampere Name of u.t Joule newton pascal watt volt coulomb ohm hertz tesla Symb M k da d c m P f = 1000 grammes = 10s grammes = O.O000000000000l grammes = Symbol . J N Pa W V C Hz T Mu/tp 10e 10 10 10-1 10-2 10101010-2 10 grammes = I000 grammes. = I000 mg = I000 lg = I000 ng = I000 pg = 1000 fg . i.) . Ci 3.) 6894.7 x 10Oserg 10-J micron () I molar.578 1 nm = lO-Sm = 10mm = 10-Tcm = 10-m Additionally $I Units for Volume The SI unit of volume is the cubic metr. llltre(1) = Idms = I0-ams Imfllflltre(ml) = Icms = I0ms I microlitre (tl) = I mms = I0 ms Conversion of Common Units into SI Unlt angstrom (A I00 pm = I0"° m = I0" em centigrade ('C) |°C + 273 } K calorie 4. Thus.s. One lltre therefore means one cubic decometre. dm.186 J cycles/sec 1 Hz curie. the terms litre and millilltre should be replaced by the terms of Sl u nits given below.76 Pa [p. ms. M ( I tool I) I tool dm gauss [G) I0 T pound-force/sq in ( Ib f In. 579 Y alpl beta gamma delta epsflon eta theta kappa lambda mu nu p! rho sgma tau phl ch! s! omega APPENDIX D Greek Symbols and Their Pronunciations . 63 97 32 65 580 BophystcoJ Chentrg Table Showing the unt of Ammonium Sulphate to be Added to 1L Solution to ..520 230 267 307 348 .313 351 251 288 189 225 158 193 127 162 12 43 74[ 107 142 31 63 94 129 31. % saturat/on Grams solid ammonium sulphate to be added to 1L of solution 209 243 277 150 183 216 29 91 123 155 301 49i 61 93 125 19' 30 62 94 390 !430 472 1516 561 [562 326]365 4061449 494i592 '300 340 1382 ... Arrive at a Particulax % Saturation Final concentrat/on of ammordum sulphate. 390i485 198 235 273 314 356 449 177 214 252 292 333 426 164 200 238 278 319 411 132 168 205 245 285 3?5 99 134 171 210 250 339 66 101 137 176 214 302 33 67 103 141 179 264 34 69 105 143 227 34 70 . 107 190 35 72 153 36.115 77 100 767 694 619 583 546 522 506 469 431 392 353 314 275 237 198 157 79 . Broad 2.86 (~ 3500} Medium 2.H) { I band} Amides (o) Primary.3.90 {3570 .79 (3650 .3590} Variable.2.2.02 (3500 .82 -2.3.340} Var/able.94 .74 . Sharp 2.94 .86 .N--H o O-H (b) Inter molecularly hydrogen bonded OH (changes on d/lut/on) (I) s/ngle bridge compounds {o} olymer assoe/aUon (¢) a moary hydrogen bonded OH-s/ngle br/dee compounds Amn (o ry. 3200} Strong.3310) Med/um 2.94 (~ 3400} Medium 2. free {2 bands} (b) Secondary.13 (3400. Shsrp 2.90 (3550 . free {2 bands} 2.3. free | I band} and Imbues |=N .03 (3400 .3450} Variable.2.3300} Medium .80 . Sharp . 3140} Medium ~ 3. bonded {2 bonds) and Atones (a) Secondary.0 .2.32 (3040 .3.92 (~ 3430} Medium .3.(b) Secondary.2 (3320 .3010} Medium 3.25 .23 .03 (~ 3300} Strong 3.3. bonded (I band} A/kene (a Monosubst/tuted } {2 bands} (b) Disubst/tuted.99 (.94 (~ 3400} ~ 2.3350} Med/um 3. free (I band} (c) Pr/msry.3075) Med/um and 3.15 {~ 3180} Med/um 3.23 .3.29 .86 (~ 3500} Medium and 2.25 {3095 . germ ~ 2. 3.3030} Var/able 3.(3095 .3.29 .3030} Medium .3 (3130 .32 (3040 .2 .38 .3075} Medium R CH = R 3.30 (.3.2853) Medium 3.3010} Medium .3.51 {2962 . 70 .2500) .2700) Weak stretching bonded.70 (2775 .3.92 (2600 .2500) Weak C--H Aldehydes (characteristic) 3.2500) Weak S--H Sulphur Compounds 3.2820) Weak stretching (2 bands) and 3.C=O stretching C=O stretching 582 Biophysical Chemistry Vbratons Absorbing Group Absorption Band Range Absorption and its Envonment (]) (cm) Intensity O--H stretclu A/coho/s and Pheno/s (a) Intra n/ecular hydrogen bonded chelate compounds 3.00 (2700 .60 .4.45 .85 .55 (2900 .1 .4.3.0 (3200 . multiple bonds 3.3. 2220) Medium a.63 .4.64 .4.2070) Medium stretching --N = C = N Dflm/des 4.2130) Medium Azdes 4.72 (2160 .42 .4.57 (2260 .46 (2275 .46 (2260 .2190) Weak stretching Variable C = N Isocyanates 4.70 (2155 .4. -Unsaturated Alkyl Nitrites 4.4.50 .51 (2235 .40 .2240) Medium Aryl Nitrites 4.Weak stretch/rig C = C A/kyne-dlsubstituted 4.50 (2240 .2215) Medium C = N Isocyankles 4.4.4.2120) Strong .83 (2220 .47 .4.2240) Medium stretching Alkyl Nitrites 4.42 .46 . 71 (1800 .53 (~ 1810) Strong (b) Acyl chlorides ~ 5.5.5. acycllc and 5. Unsaturated and 5.5.57 (~ 1795) Strong .35 .41 .62 (1830.1180) Weak Anhydrales (a) Saturated 5-membered 5.1820) Strong ring and 5.65 .47 .1780) Strong (c) Saturated.56 (1850 .and 7.1750) Strong (b) a.41 . acyclic 5.1780) Strong aryl.5.59 .1800) Strong and 5.46 .5.56 . -Unsaturated 55.48 (1340 .47 .1800) Strong membered ring and 5.5.49 (1870 .62 (1830 .5.75 (1790 .1720) Strong Acyl Halldes (a) Acyl bromides ~ 5.56 (1850 .5.8.1740) Strong (d) a.81 (1770 . 41 (~ 1850) Strong (d) a.5 (~ 1820) Strong 5.5.72 (1780 .72 .62 .!750) Strong and aryl and 5.68 (1780 .1760) Strong .1720) Medium Esters (a) Saturated. -Unsaturated 5.(c) Acyl fluorides ~ 5. cyclic (I) [3-]actones ~ 5.61 .82 (1750 .5.5. 78 .5.71 .1717) .76 (1750 .1735) Strong (c) Unsaturated (I) Vinyl ester type 5.76 (1750 .5.1735) Strong (b) Saturated.82 (1730 .C=O stretching C=O stretching C=O stretching = 0 C=N stretdUn C=O stretching C--O stretchg Vibrations Absorbing Group Absorption Band Range Absorption and its Environment . () (cm) Intensity (3J 6-1actoncs (and larger rings) 5. B-unsaturated and aryl 5.65 (1800 . acyclic 5.56 .5.5.1770) Strong (2) a.71 . 71 .1695) Strong (c) a.87 .81 (1740 .75 .5. aliphatlc 5.every aldehyde has characteristic C m H stretching vibrations (2 bands} at 3. acycllc 5.2700) Weak Ketones (a) Saturated. cyclic (I) 6-membered ring (or higher) 5.1740) Strong (a) Saturated.1680) Strong Note : .87 (1725 .5.80 .5. -Unsaturated. allphatic 5.5.3.90 (1715 .1720) Strong (b) Aryl 5.75 .1705} Strong (b) Saturated.45 .55 (2900 .70 (2775 .2820) Weak and 3.87 (1725 .5.62 .Strong (d) Carbonates 5.83 .60 .1"705) Strong (2) 5-membered ring 5.5.80 .95 (1705 .3.75 (1780 .5. 1708) Strong (e) Aryl 5. aliphatic 5.5.85 (1724 .5.92 (~ 1690) Strong (b) Secondary.1665) Strong (2) 5-membered ring 5.1680) Strong Amides (a) Primary.99 .1670) Strong . dilute solution ~ 5.80 . cyclic (1) 6-membered ring 5.6.88 .94 .5.88 (1725 .6.95 (1700 .80 .1740) Strong (c) a. dilute solution 5.1700) Strong (b) a.01 (1685 .1685) Strong (d) a. acyclic 5.68 .02 (1670 .94 .92 (1715 .95 (1700 .1680) Strong Diaryl 5.1690) Strong (c) Aryl " 5.(1750 .6. B-Unsaturated.88 .5. aliphatic 5. B-Unsaturated.5.5.83 .01 [1685 .1660) Strong CarboxI/c Adds (a) Saturated. B-Unsaturated.99 (1700 . 10 (1690 .Imlnes.92 . Oximes (a) Alkyl compounds 5.1650) Strong .6.1540) Strong Esters (a) -ketoesters (enolic) ~ 6.06 (.1640) Strong Ketones -Diketone 6.50 (1640 .10 .6. 21 .69 (1400.6.1550) Strong stretchin and 7.7.NO2 stretchln CmNO setch/ng (b) Carboxylate anion 6.1300) Strong Amides (a) Primary.C=O C=C stretch/ng NmH C = NO2 stretching (b) Aromatic Nitrates Noso Co 584 Biophysical Chemistry Vibratfons Absorbing Group Absorption Band Range Absorption and fts Environment . solid and concentrated solution .45 (1610 . () (cm) Intensity 0.15. 6.97 (~ 1675] Medium (e) Disubstituted.6.17 (1680 .~ 6.29 (1650 . trams ~ 5.95 .1620) Variable (b) Monosubstituted (Vinyl) .1590) .1620) Strong Alknes (oJ NonconJugated 5. cfs .6. solid and concentrated solution 5.06 (~ 1650) Strong .99 (~ 1669) Medium (9) Tetrasubstituted ~ 5.99 .99 (. gem ~ 6.14 (1680 . solid and all solution 5. (b) Secondary.03 (~ 1658) Medium (d) Disubstituted.08 (.1630) Strong (c) Tertiary.1645) Medium (c) Disubstltuted.17 (1680 .95 .6.6.06 .1669) Weak Amfnes (a) Primary 6.6.05 (~ 1653) Medium (]) Trisubstituted ~ 5. 30 (1380 .45 (1570 .1550) Weak (c) Amine salts 6.37 .25 .14 .6.63 (~ 1580) Variable vibrations) ~ 6.6.17 .35 (1630 1575) Variable N/tro Compounds (a) Aliphatic 6.25 .Strong (b) Secondary 6.29 (1620 .67 (~ 1500) Strong Amides (a) Primary.06 .90 (~ 1450) Medium Azo Compounds 6.1590) Strong Aromatfcs (4 characteristic bands due to ~ 6.6.90 (~ 1500) Medium ~ 6.1370} .25 (~ 1600) Variable skeletal carbon stretching ~ 6.6. dilute solutions 6.7.6.35 (1600 .1550} Strong and 7.1575) Strong and ~ 6.45 (1650 . 00 (1300 .7.70 o 8.25 (1650 .Strong 6.1300} Strong 6.6.30 .6.1500} Strong and 7.67 (1570 .06 .25 .37 .1250) Strong 6.6.67 (1600 .1600} Strong and 7.1500) Strong .70 (1370 . shorter wavelength due to . () (cr) Intensity Nitrites 5.C---H O--H (d} Phenols O--NO sretch/ng N--H Appendices 585 Vrtons Absorbing Group Absorption Band Range AbsorptWn and its Environment .95 .06 [1680 -i650) Strong Amkles (a) Secondary amides (dilute solution) Alkanes (oJ --CH=-.6.(e) C--H A/coho[s and Phenols Two bands .(scissoring] (b) C--CH (c) Gem dimethyl (isopropyl) (d Teary-butyl . 1510} (t385.33 7.1430) {1380.74 .62 (1550 .7.25 .7.1340) Medium .00 7.17 .7.30 7.80 .22 .6.33 7.1510) Strong 6.7.92 6.25 7.22 .7.45 .30 .1445) (1470.O--H bending and longer wavelength band caused by characteristic C--OH stretching vibration (a) Primary alcohols (b) Secondary alcohols (c) Tertiary alcohols and and and and and and 6.6.7.46 (1485.1380} (]370 1365) (1395" 1385) (~ 1365) (. 7.4 .9.93 .9 8.55 .05 7.6 8.4 .1 .I010) (1350.7.30.9 9.77 (1350.90 7.1260] (I 120 .7.13 .1140) Strong Strong Strong Strong Strong Strong Strong Strong C--O--H stretching C--O and OH Aromatic out of plane .09 7.Medium Strong Strong Strong Medium Strong Weak 7.1310) {1230 .1 .9.I I00) (1410.7.I I05] (1410.8.1260} {1075.6 8.1310) (I 170 .9. 7.{a) Unsaturated.87 .70 .8.28 (1320 1210} 6.8. Strong .58 . 1250 cm-j (b) OH bending Aomatics Substitution Type (a) 5 adjacent hydrogens 7. Aryl (b) Aliphatic Carboxyl/c Adds (a) C--OH stretching (usually characteristic doublet near 8.13 {1270 1230) 8.95 .43 {I150 I060} 7.17 {14401395) ~ 13.3 (~ 750} {not always present} Strong Strong Strong Weak Variable.9.00 . 8 (.(dlsubstituted ~ 13.600) Variable.8 (~ 780} k: DisubUtuted.6 (800 .3 (~ 750) (c) 3 adjacent hydrogens (trisubstituted} ~ 12. Medium Strong Strong Any compound where n>4 ~ 13. Strong Var/able..5 (.16.. cis ~ 14.722} Strong .5 .690} Ch-o 12.3 {~ 700} (always present} (b) 4 adjacent hydrogens .14. Gel Filtration Chromtography Protein s Moleculor weight Aprotlnin from bovine lung Cytochrome C from horse heart Carbonic anhydrase from bovine erth ocytes Albumin from bovine serum Alcohol dehydrogenase from yeast -amylase from sweet potato Apoferritln from horse spleen Bovine thyroglobulln Blue dextran 6.000 150.587 Appendices APPENDIX G Commonly Ued Molecular Weight Markers for Protein 1. SDS Gel Eletrophoresis Mo/ecu/ar we@hi a-Lactalbumin -Lactoglobulin Trypsin inhibitor from soyabean .000 443.400 29.000 200.000 66.000 2000.000 669.500 12.000 2. alactosidase from E.Trypsinogen. ot.400 20 I00 24.000 272..000 Several other proteins. have also been used as molecular we/ght m arkers.co/t a2-macroglobulin from human plasma Urease from Jack bean 14.400 116 O00 180. PMSF treated Carbonic anhydrase from bovine erythrocytes Pepsin Egg albumin Pyruvate kinase from chicken muscle Bovine albumin Fructose-6-phosphate kinase from rabbit muscle Phosphorylase b from rabbit muscle -g.200 18. .000 29 000 34 000 45 000 58 000 66 97 .cited here. 588 1. ¥ prays .1 0.and particles 6Zn 82Br mSr OSr 1311 8121.4.1 days 8-.85 APPENDIX H Radioisotopes Frequently Used as Tracers and Their Properties Radioisotope Type of Half-life Energy of radltion.3 days 887. MeV Radiation a .6yr 8-. 2. ¥ 14.8 hr 814.1 yr 857oo yr 8% ¥ 2. 0 (25/) 0. 154 0.57 (8-) 0.26 0.66 (8+} 0.4 1. Y 47 days 8-. 8-.31 0.12.46 0. y 250 days 8-.58 1.7 0.¥ 5.017 O.4 hr 8180 days ¥ 310 days x. 0. ¥ 34 hr 855 days 825yr 8-.3yr X-ray.26.32 . ¥ 8 days 0.17 2. 8÷ 12.8 hr ¥ 8÷.y 8-. 1.3 1.465 1.0.35 I.79.35 0.7 I.I 0.08.I.55.6 1.38.32 1. 0.16.5 0. 1.6 0. 1. 2.3 1. 0.65 .37. 0. Actinium Alumlnlum Antimony Argon Arsenic Barium Beryllium Bismuth Boron Bromine Cadmium Calcium Carbon Ceslum Chlorine Chromium Cobalt Copper Europium Fluorine Gallium Gold Helium Hydrogen Indium Iridium Iron Krypton Lanthanum Lead Lithium Magnesium Ac Al Sb Ar As B! B Br . Cd Ca C Cs Cl Cr Co Cu Eu F Ga Au He H In Ir I Fe L! Mg 89 13 51 . 18 33 56 4 83 5 35 48 20 6 55 17 24 27 29 63 9 31 79 2 1 49 77 53 . 95 74.34 9.75 39.72 196.99 69.92 137.01 132.08 12.96 18.99 58.98 121.81 79.93 151.35 54.96 .9 112.26 36 57 82 3 12 Atonrlc Weight 227 26.98 10.91 35.01 208.4 40. 9 55.8 138.4 1 118.94 24.82 192.3 589 Table of Atomic Weights and Atomic Numbers Symbol Atomic Number .85 83.22 126.2 6.9 207. 59O Element Symbol Manganese Mercury Molybdenum Neon Nickel Nitrogen Osmium Oxygen Palladium Platinum Plutonium Polonium Potassium Radium Radon Rubidium Ruthenium Selenium Silicon Silver Sodium Strontium Sulfur Tellurium Thallium Thorium Tungsten Uranium Vanadium Xenon Yttrium Zinc Zirconium Mo Ne Ni N Os O Pd Pt Pu Po K . Ra Rn Rb Ru Se Si Na Sr S Te Sn U V Xe Y Zn Zr 25 8O 42 10 28 7 76 8 46 78 94 84 19 88 . 94 20.71 .17 58.93 200.59 95.86 37 44 34 14 47 11 38 16 52 81 90 5O 74 92 23 54 39 3O 4O 54. 14.02 222 85.1 226.0 190.02 50.85 238.2 16.98 87.09 242 210 39.4 195.96 28.62 32.03 118.0 106.37 91.O8 107.6 204.07 78.3 88.06 127.46 101.69 183.86 22.9 65.37 232.94 131.22 Biophysical Chemistry Atomic Number Atomic Wejht . %T E %T E %T E %T E lO0 0.013 72 0.131 49 0.004 .143 47 0.602 99 0.328 22 .30l 25 0.000 75 0.125 50 0.620 98 0.319 23 0.009 73 0.591 Appendices APPENDIX J Trmsmission/Extinction Itelatlonship between the transmission in % ( %T ) and the extinction ( E ) ( absorbance.638 97 0.137 48 0.310 24 0. optical density ).74 0. 337 21 0.161 44 0.770 91 0.181 41 0.721 93 0.699 94 0.027 69 0.658 96 0.036 67 0174 42 0.187 .168 43 0.022 70 0.357 19 0.387 16 0.678 95 0.046 65 0.155 45 0.367 18 0.149 46 0.041 66 0.377 17 0.0.018 71 0.796 90 0.032 68 0.745 92 0.347 20 0. 208 37 0.056 63 0.824 89 0.215 36 0.000 84 0.071 60 0.076 59 0.229 34 0.432 12 0.061 62 0.959 85 0.398 15 0.444 11 0.409 14 0.886 87 0.194 39 0.051 64 0.046 83 .201 38 0.921 86 0.066 61 0.40 0.222 35 0.420 13 0.456 10 1.854 88 0.469 9 1. 509 6 1.237 33 0.108 53 0.523 5 1.086 57 0.398 78 0.301 79 0.523 7 0.495 7 1.552 3 1.538 4 1.276 28 0.155 81 0.569 2 .102 54 0.097 82 0.244 32 0.252 31 0.222 80 0.284 27 0.0.114 52 0.097 55 0.092 56 0.260 30 0.268 29 0.482 8 1.081 58 0. 699 76 0.119 51 0.000 .292 26 0.585 I 2.1. 160. #01 Affinity precipitation.& Absorbance. 191 orientation effect. 161 and Van der Waals forces. 159 and induction forces. 160 from solutions. 206 Acid-base catalysis. 158 characteristics of. 157(I) Adsorbent. 191 Absorption spectra. 397-398 variations of. 438 Alpha emission. 396-404 applications. (see polyacrylamide) Activity coefficient. 401-402 Ag/AgCI reference electrode. 364-365 Affinity chromatography. (see extinctioncoefficient) Absorption maxima. 158 and hydrogen bonds. 403-404 isolation of DNA-binding proteins. 403 lignd selection. 157-174 and dispersion forces. 380 ( also see gels) Agarose-acrylamide. 186. 187 factors affecting. 190-192 effect ofpH. 481 . 398 ligad etachment. 57-58 Aarose. 190(I) effect of polarity. 49 Acrylamide. 404-405 supporting matrix. 178 Absorption coefficient. 159 and molecular orientation. 48. 349. 172-173 Adsorption chromatography. 201-205 interpretation. 201-205 Absorption spectrum. 157(i Adsorption. 9 Adsorbate.purification of mRNA. 47 Acid-base properties of hemoglobin. 203-205 structural analysis based on. 171-172 importance of. 401-402 Affinity cross-flow ultraffltrtion. 402 standard matrix-ligand systems. 187-190 Absorptivity. 186-191 electronic transition and. 398-400 . 179. Alpha particles. 321-342 Analytical ultracentrifuge. 457 (also see isoelectric focussing) Ampholytes. 500 b-mercaptoethanol. 307(i) Anion exchanger. #84-485 Ascending chromatography. 186 Beer-Lambert law. 184 isobestic point. 185 turbidity. 334-339 Bend-shift assay. 186 spectral shift. 487 Amicrons. 183-186 fluorescence. 495 Auxochrome. 352 Association colioids. 210. 426. 36 Ampholine. (see gel retardation) Bathochromic shill. 84 Atomic absorption spectrophotometry. 83. 457 (also see isoelectric focussing) Analytical centrifugation. 177-186 deviations from. 178 Bending vibrations. 436 Amphiprotic compomds. 389. 390. 71 (also see ion selective electrodes) Ammonium persulphate. 247 Atomizers. 439 Band sedimentation. (see nebulizers) Autoradiography. 185 instrumentation limitations. 220 Bequerel.392 Archibald method. 484 . 184 sample instability. 393-394 Ammonium ion electrode. 192 B b-energy spectrum. 306-308. 186 temperature effects. 480. 484 production of. 76 Amino acid analyser. 192(i Beer's law. 184 imperfect monochromacy. 342 Arrhenius theory. 391. 2-3" Artificial isotopes. 245 Atomic fluorescence. 30 glycylglycine buffer. (also see luminometry) Biosensors. 457. 241 Birefringence (see flow birefringence) Blank. 56-57 Capillary flow. 28-29 Buffering of blood. 188-190 Bouger-Beer law. 367. 27910 . 23-31 bicarbonate buffers. applications of. 279-280. 73(f) Carboxymethylcellulose. 29 EDTA buffers.. 178 Boundary sedimentation. 240. 390. 48 carbnonate buffers. 178 Bouger-Lambert law. 390 Carrier ampholytes. 30 Good buffers. 131-132 Carbon dioxide electrode. 298 Bronsted-Lowry theory. 366. 533 Bioluminescence.Carrier gas. measurement of viscosity by.392 CD/ORD spectra. 195 Bohr effect. 330-334 Bragg equation. 274-276 of carbohydrates. 30 tris buffers. 182 Blue-shift. 73-74. 3-6 Buffer capacity. 368 Cation exchanger. 398. 48 Bonding/antibonding orbitals. 48-50 Buffers. 459 (also see isoelectric focussing) . 30 glycine buffers. 29-30 Calomel reference electrode. 29 triethanolamine buffer. 177.594 Biological half-life. 30-31 phosphate buffers. 391. 404 Circular dichroism. 305-311 in zonal rotors. 360-361 Column electrophoresis. (see. 122 Colloidal osmotic. 148(i . 502 Chiral chromatography. 488 Conjugate acid/base. 271-283 instrumentation for. 498-499 Chemical shifts.442(f} Compton scattering. 361-363 flow rate. pressure. 359-364 columns.367 Chromatography. survey of. 76 properties of. 283 CM-cellulose. carboxymethylcellulose) Coefficient of viscosity. 352. 241 Chemiluminescent substances. 275-278. 281 If) or'proteins. 280(0. 361 sample introduction. 87-92 Color quenching. 280-282.192 Cibacron-blue-agarose. 49 Chromatogram. 504 Channels ratio. 301-343 instrumentation. 418 Chloride shift. 75 classification of. 499 Column chromatography. 344-421 procedures. 359 elution. 463-464 Cellulose acetate elcctrophoresis. 120.278(0 Cellular gel. 147-149. 440-442. Colloids.of nucleic acids. 240 Chemiluminescent assays. 500-501 (also see scintillation counting) Biophysical Chemistry Chemical quenching. 277(0. 361 effluent collection. 319-321 Cerenkov counting. 346-350 Chromophore concept. 78-80 definition of. 251-253 Chemiluminescence. 19 I. 5 Contact angle. 435 Centrifugation. 286-287 CTAB. 317 Continuous discharge. 133 . 147. 381 Couette rotating-cylinder viscometer. 419 Craig apparatus.of bacteria with neutrophils. 347-348 Counting efficiency. 490 Controlled pore glass beads. 439 .134. 147-149 and phagocytosis. 148 Continuous density gradient. 348 Cross of isocline. (see under scintillation counting) Covalent chromatography. 133( Countercurrent distribution. 403 DNA-cellulose chromatography. 448-451. application of. 445 Difference spectroscopy. 316-317 rotors. 482. 378-379 Diamagnetic shielding. 305 Dextran gels. 417-418 DNA footprinting. choice of. 318 sample. 449 stacking gel. 444 Density gradient centrifugation. 101 significance of. (see discontinuous gel electrophoresis) Discontinuous density gradient. 450 Disintegration constant.320 density gradients. 106 Distribution coefficient. 499 Dimethyl POPOP. 251. 319 sample. 208 Differential centrifugation. 377 DNA-affinity chromatography. 158 Dissymetry constant. 390 Decay constant.Index Curie. 317 318 gradient materials. 106-109 Diffusion potentials. 100-109 Diffusion coefficient. 472-473 . 318-319 Desk top centrifuges. 483. 432 Dead time. 103-106 Diffusion of electrolytes. 317 Discontinuous gel electrophoresis. recovery of. 485 D Danieli and Davson model. 313-317.252. preparation of. 195(1) Diffusion. 318. 484 Densitometry. 153-154 Dansyl chloride. 31i-313 Diffraction grating. 319. 497 Disc gel electrophoresis. 253 Dideoxynucleotide sequencing technique. 484 Cyclotron. 108 Dilution quenching. 449{0 separating gel. 492 DEAE-cellulose. 482 Dispersion forces. 200(I) Dual isotope analysis. 1-2 Electromagnetic spectrum. 52-54 Electroendosmosis. 90(0 Electrophoresis. 549-550 Donnan effect. 438 Stoke's equation. 533 thermoluminescence dosimetry. 70 Electrode potential. i-2 Electrolytic dissociation. 176-177. 423-424 in gels. 425 sample. 423-426 buffer. 63 Electrode interference. (also. 426-430 on stabilizing media. 534 Double-beam spectrophotometer. 423-426 factors affecting. 201(1) EDTA buffers. 533 Electric birefringence. (see Zone electro phoresis} Electrophoretic mobility. 468 Electrode contamination. 469-472 DNA thermal cycler. 503-504 Dual wavelength spectrophotometer. 79 Donnan equilibrium.DNA sequencing. 258-261 applications of. 425-426 electric field. 424-425 medium. 95-99 biological significance of. 422-476 types of. 90 Electrofocussing. 98-99 Dosimetry. 258(1) Electrosmosis. EC decay). 533-534 Fricke dosimeter. 200-201. 177(1) Electron capture. 289 Electrical double layer. 87 E1ectroblotting. 30 Effective half-life. 481 Electron spin resonance spectrometry. (see isoelectric focussing) Electrolytes. 259-261 instrumentation for. 533-534 film dosimeter. 423 . Electroviscous effect. 481 . 558-560 Elliptical polarization. 272 Emission of gamma rays. 130 ELISA. 500 Extinction coefficient. 134-136. 242-247 nebulizers. 516 (also see labeling formats) Ethidium bromide staining. 11. 515. 164 Emulsoids. 71 Enzyme-linked immunosorbent assay. 243-244. 284-289 . 179 Extrinsic fluorescence. 12-14 Equilibrium isotope effect. 381 External standardization. 101-102 Field inversion gel electrophoresis. 463 Flame ionization detector. 432. 165 ion antagonism in. 228 F Facilitated diffusion. 505-506 Fick's laws of diffusion. 134() Falling drop method. 71(f} ammonium electrode. 162-165 HLB number and. 371-372 Flame sPectrophotometry. 163 inversion of.596 Emulsions. 244(1} Flocculation value. 441 Exchanger capacity. 510 Equilibrium labeling. 79 Enzyme assay. 179-182 molar extinction coefficient. 100 Falling ball viscometer. 165 oil in water. 164 water in oil. (see ELISA) Equilibrium constant. 392 Exclusion chromatography (see gel permeation) Exclusion limit. 377. of gels. 92 Flow birefringence. 207 Enzyme electrode. 562-563 Fluidity. 235-239 Fluorescent dyes. 232-233. 366-375 applications. 285-286 Flow cytometry. 3S8 Fluorescence ene ta'nsfe. 222 Fluorescence. 123 Fluorescence. 560-565 applications. 221-223 Stolce¢ shift. 234(I} tRNA structure. 368 detectors. 3?4-375 carrier gas. 371-373 liquid phase. effect of. 235 enz3nne conformation. 223(1} Fluorescence polarization. 367 . 433 Fluorescent probes. changes in. studies on. 495 Fraction collectors. 232.176 Frictional ratio.apparatus for. 215 Gas flow counters. 233-234. 370 retention volume. 505 Free electrophoresis. 226 Fluorescent staining. 426-428 Frequency. 487 Gas cells. 564-565 instrumentation. 362-363 Fragment long. 222(] structural factors. 106 Gamma rays. 228-235 applications of.. 221-222.. 175. 235 Biophysical Chemistry pyruvate dehydrogenase complex. 493 Gas-liquid chromatography. 444 Fluorometry (see spectrofluorimetry] Fluors. 494 continuous discharge. 435-448 applications of. 491-494 background count. 368-370 Gas-sensing electrodes. el ze at/on . 71-73 Gas solid chromatography. 492-493. 493(1} dead time. 440-443 molecular weight. 493 Gel electrophoresis. 438-439 Gel Itrar'on claromatoaphy. 445 procedv-re oxr. 492 self absorption.solid support. 439-440 soIub'Iizers. 445-447 detection. 443-445 modes of.chromatography} . determination by. recovery and estimation. 492 Geiger region. 87 Geiger counters. 374-375 Gegenions. 494 tube characteristics. (see. 447 of nucleic acids. 492 counter tubes. 512(I) geometric effect. 494 quenching. 438 polyacrylamide 436-437. 24-27 Heparin-agarose. 436 agarose-acrylamlde. 437(t} starch gel. 2 I0 . 436 Gel-sol transformations. 473-476 Gelatin filters. 143 Gene cloning.Index Gel permeation chromatography. 30-31 Gradient gel electrophoresis. 550-551 Genetic engineering. 194 Gels. 58-62 Glass membrane electrodes. 94 Hooke's law. 436-438 agar. 168-171 Glass electrode. 378-381 Gel retardation electrophoresis. 383-385 molecular weight determination by. 483 Helmholtz double layer model. 82-83. 404 Hess viscometer. 87(fl Henderson-Hasselbalch equation. 494 Gibbs' adsorption equation. 76{i Good buffers. 306 High voltage electrophoresis. 31 Graphite rod atomizer. 163 Hofmeister series. 405416 High speed centrifuges. 244-245 Gyromagnetic ratio. 67-68 Globar. 132-133 High performance liquid chromatography. 383-385 types of gels. 453(i HLB number. 193 Gold sol curves. 241 Geometric effect. 452 Gradient maker. 249 H Half-life. 385 applications. 382 analytical uses of. 452-453. 376-386 advantages. 123 Inverse PCR. 386-395 applications. 460-461 Immunoradiometry (IRA). 210 vibrational frequencies. 58 Hydroxyapatite chromatography. 254-256 Hypochromlc effect. 135 Hydrogen electrode.HPLC. 412-416 ion chromatography. 192(i Hyperfine splitting. ion pairing. 192(i I Immunoelectrophoresis. 213-214 of carbonyl grOuP. 219 modes of vibration. 410-411 pumping systems. 499-500 Intrinsic viscosity. 212-215 of carbon skeleton. 216-220 bending vibration. 209-220 applications of. 212 infrared dichroism. 210 fingerprint region. 211-212 overtone bands. 528-529 Imperfect monochromacy. 409(i guard column. selection of. 414-416 ion suppression. 393-394 . 215-216 stretching vibration. 411(fl reverse phase chromatography. 412-413. 409 597 separation system. 54-56 Hydrogen ion activity. 185 Infrared gas cells. 214 Infrared spectrophotometer. 153-154 Internal standardization. 413 Huggin's plot. 409-410. 414-416 Ion-exchange chromatography. 407-409 sample injection. 214 of nitrogen compounds. 147. 215 Infrared spectroscopy. (see high performance liquid chromato graphy} column packings. 165 Ion chromatography. 196 Infrared spectra. 211 sampling techniques. 416-417 Hyperchromic effect. 210211 Interfaclal tension. 411-412. 214 of hydroxy compounds. 552 Ion antagonism. 192(f) Hypsochromic effect. 123(t). calcttlation of. 391 ion-exchange resins. 392-393 . 387 exchange medium.counterlons. preparation of. 389-390 procedure. types of. 42-43 of strong acids. 11-12 of weak acids. 519(i9 fate of cells. 413 Ion product of water. 412-413 Ionization. 12-14 Irreversible colloids. determination of. 518-530 clinical applications. 453-460. 70 method interference. 335. purification of. 38 Isopycnic centrifugation) 314. 44 of protein. 489. separation of. 13 Ion-specific electrodes. 66-74 basic principles. 524-525 mechanism of enzyme action.598 Ion-pairing. 457 Isoelectric pH. 47 Isotope exchange studies. 479 definition of. 490 Ionization constant of water. 17-19 dissociation constant. 18 Ionization chambers. 518-519. of amino acids. 93 Isobestic point. 519-521 Isotope effect. 336 nucleic acids. 336 Isotope. 70 applications. 315. 36-41 of proteins. uses of. 522-523 Isotope incorporation. 524-525 . 522 Isotopes. 184 Isoelectric focussing. 455 Isoionic point. 20 of water. 70-71 Ion suppression. 521-522 isotope incorporation/exchange studies. 44 Isoelectric point. 335 plasmid. 525 distribution studies. 6667 electrode interference. 479 Isotope dilution. 454(f) carrier ampholytes. 130 Kronig-Kramer transform. 515-516 pulse labeling. 524 mineral metabolism. 79 Lyophobic colloids. 10-11 Laws of absorption. 166(f) Lanthanum fluoride electrode. 69(t] Luminometry. 293 Law of mass action.membrane transport studies. 275 L Labeled compounds. 365-366 Liquid-membrane electrodes. 290-292 Limiting viscosity number. 4 Light scattering. 525 study of protein structure. 153154. 103 Laue pattern. 405 Lewis acids and bases. 513-517 equilibrium labeling. 404 M Mass spectrometry. 524 sterilization of foods. 219 Liquid-liquid chromatography. 469-472 Membrane potential. 241 Lyophilic colloids. 162 . 529-530 Jhonston'Ogston effect. synthesis of. 226 Membranes. 506-508 Krasny-Ergen equation. 513-515 pulse-chase labeling. 505(f) Matrixless afl'mity separations. 79-80 Lysine-agarose. 510 Knoop's experiment. 165-166. 485-487 Labeling formats. 69-70. 521 metabolic studies. 123 Linear dichroism. 177-186 Lectin-sepharose. 68 Lattice theory. 523-524 metabolic turnover time determination. 402 Maxam-Gilbert DNA sequencing. 155 Kinetic isotope effect. 504-505.517 LanEmuir trough. 240-242 Lux genes. 516. 322-323 Biophysical Chemistry K Kelvin equation. Metabolic turnover time. 84-85 Microelectrophoresis. 403-404 Method interference. 76 Miller indices. 426-427 Microns. 296 Molar ellipticity. 274 . 70 Micelles. 524 Metal chelate affinity chromatography. 339-340 by X-ray diffraction. 506 Northern blotting. 120 Optical density. 271-283 . 481 Negatrons. 115 O Oncotic presure. 249-251 Number average molecular weight. 207-208. 178 Optical quenching. 160-171 in emulsions.Index Molecular bending. 256(f) Nuclear resonance. 447 by gel permeation chromatography. 194 Monolayers. 176 Molecular orientation. 168(i] Mossbauer spectroscopy. 53 Nernst glower. 247-258 applications of. 261-264 Moving boundary electrophoresis. 466 Nuclear magnetic resonance spectrometry. 256-258 instrumentation for. 499 Optics/rotation. 383-385. 427(0 N Negatron emission. 271 Optical rotatory dislrsion. 193 Nitrate electrode. 339342. 342 by gel electrophoresis. 165-168 of steric acid. 341-342 by sedimentation velocity. 299-300 Monochromators. 71 Non-radioactive labeling. 447 by absorption spectrophotometry. 209{i] Nernst equation. 425-426. 383-385 by osmotic pressure measurement. 114-116 by sedimentation equilibrium. 487 Nephelometry. 207-208 by approach to equilibrium. 162-165 in membranes. 427-48. 162 in monolayers. 165-168 Molecular weight determination. 51-62 scale of. 430-435. 73(I) Pair production. 52-62 measurement of. 113 isosmotic. 344-345 Passive diffusion. (see protective pH. 120 definition of. 346-349 Partition coefficient. 488 Paper chromatography. 62-64 . 117 Osmoregulation. 1 I0. (see polymerase chain reaction) Peptizing colloids. 15 pH meter. 352-5 ascending technique. 109-121 definition. 117-121 excretory systems and. 352-353 descending technique.instrumentation for. optical rotatory dispersion) Osmolarity. 119-120 Osmosis. 113 Osmoreceptors. 431(f) apparatus. 11 measurement of. 109 electronic osmometer. 282-283 ORD (see. 116-121 Osmotic pressure colloid osmotic pressure. 430-432 Partition chromatography. 110-112 total osmotic pressure. 112-114 significance in biology. 177 PCR. 14-17 clcctromctric determination of. concept of. 352-353 detection. 100 Path-length. 461 Oxygen electrode. I i0 Ouchterlony method. 354-355 solvent. 430 detection and quantitative assay. development of. 353-354 Paper electrophoresis. 351-357 apparatus. 114 hyperosmotic. 352-353 paper. 432-34 elcctrophoretic run. 113 hyposmotic. 109 osmotic behaviour of cells. 72-73. choice of. 197-198. 198 Photoelectric absorption.pH paper. 197(t] Photomultipliers. 35 . 497 Photodiodes. 223. 197(f) Physiological buffers. 198(f) Photovoltaic cells. 488 Photoemissive tubes. 64 Phosphorescence. 196-197. 114 Viscomctry. 140 native structures of proteins. 175 . 195-196 wavelength selectors. 137-138 size and shape of macromolecules. 141 intrastrand disulphide bonds. 140 protein denaturation. 177(f}. 131-136 of ellipsoids. 125-126 of spheres. 311 Water regain value. 378 Wave number. 142-143 factors affecting. design of. 206 instrumentation for. 136 applications of. 377(f} Wall effects. 310. 193 sample containers. 377-378. 136137 Viscornetcr. 138 DNA lincarity. 122-144 biological significance of. 19--201 detection devices. 143 measurement of. 136-141 actin-myosin interaction. 132-133 Biophysical Chemistry Viscosity. 175 Wavelength. 128-131 hematocdt and.602 quantitative analysis. 138-139 association-dissociation studies. 124 Void volume. 193-195 Vacuum ultraviolet region. 190 van Hofra equation. 125 of random coils.. 141-143 blood flow and. detection of. 140-141 Intercalation of dyes. 141 DNA polymerization. 196-198 radiation sources. 138 chemical modifications. 468-459 X-ray diffraction. 90-92 Zonal centrifugation.Western blotting. 293-300 Z Zeta potential. 319 Zone electrophoresis. 88-92 measurement of. 428-430 .