Laboratory Manual forCHEMISTRY 315/325/335/345 Department of Chemistry University of British Columbia 2013-2014 I I How to cite this manual: Laboratory Manual for Chemistry 315/325/335/345; Bussiere, G., Kunz, T., Monga, V., Rogers, C., Stoodley, R., Eds.; University of British Columbia: Vancouver, Canada, 2013 I I I TABLE OF CONTENTS THIRD YEAR CHEMISTRY LABORATORY LEARNING GOALS ................................. VI LABORATORY DIRECTOR CONTACT INFORMATION .................................................................... X YOUR PERSONAL LABORATORY SCHEDULE ............................................................................. XII LABORATORY LOGISTICS ......................................................................................................... XIV DURING THE LAB SESSION........................................................................................................ XVI LAB REPORTS .......................................................................................................................... XVII ACADEMIC HONESTY ............................................................................................................. XVIII SAFETY INSTRUCTIONS ............................................................................................................. XIX SAFETY EDUCATION/TRAINING PROGRAM ................................................................................ XX WORKING SAFELY IN CHEMISTRY LABORATORIES ................................................................ XXI EMERGENCY AND NON-EMERGENCY PHONE NUMBERS ....................................................... XXII HAZARD SYMBOLS AND CLASSES .......................................................................................... XXIII ANALYTICAL CHEMISTRY EXPERIMENTS .............................................. 24 ANALYTICAL CHEMISTRY LABORATORY COURSE ..................................................................... 25 ANALYTICAL GRADING STRUCTURE ........................................................................................... 26 PREPARATION FOR AND DURING THE LAB .................................................................................. 28 ANALYTICAL CHEMISTRY REPORT FORMAT .............................................................................. 29 ACCOUNTING FOR UNCERTAINTY ............................................................................................... 31 A-03 DETERMINATION OF LEAD BY ANODIC STRIPPING VOLTAMMETRY ................................ 40 A-04 DETERMINATION OF IRON AND CHROMIUM BY SPECTROPHOTOMETRY .......................... 44 A-06 DETERMINATION OF QUININE BY FLUOROMETRY AND ABSORBANCE SPECTROMETRY .. 49 A-08 DETERMINATION OF CR 3+ BY CHEMILUMINOMETRY ........................................................ 65 A-09 DETERMINATION OF CALCIUM IN THE PRESENCE OF ALUMINUM BY ATOMIC ABSORPTION SPECTROMETRY ..................................................................................................... 72 A-11 DETERMINATION OF COPPER BY ICP-MS – ISOTOPE DILUTION MS ............................... 78 A-12 DETERMINATION OF MANGANESE BY NEUTRON ACTIVATION ANALYSIS ........................ 88 A-13 DETERMINATION OF NITRITE AND NITRATE BY ION CHROMATOGRAPHY ....................... 97 A-14 DETERMINATION OF BIPHENYL AND P-TERPHENYL BY LIQUID CHROMATOGRAPHY ... 103 A-16 DETERMINATION OF NAPHTHALENE BY GC-MS ............................................................. 113 INORGANIC CHEMISTRY EXPERIMENTS ............................................... 122 EXPERIMENTS ASSIGNMENT AND LABORATORY ROUTINES .................................................... 123 INORGANIC GRADING STRUCTURE ............................................................................................ 125 INORGANIC CHEMISTRY REPORT FORMAT .............................................................................. 126 I-02A SYNTHESIS AND CHARACTERIZATION OF A DIPHOS LIGAND ......................................... 128 I-02B PREPARATION AND ANALYSIS OF SOME NI(II) DIPHOS COMPLEXES ............................ 137 I-03A PREPARATION OF TETRAETHYLTIN ................................................................................ 145 I-03B REACTION OF TETRAETHYLTIN ...................................................................................... 153 I-06 PREPARATION AND MAGNETISM OF CHROMIUM(II) ACETATE ........................................ 159 I-07 STUDY OF CU(II) AMMINE COMPLEXES ............................................................................ 165 I-10A ELECTRO-SYNTHESIS OF VANADIUM(III) ALUM ........................................................... 173 I-10B DETERMINATION OF THE COMPOSITION OF V(III) ALUM ............................................. 180 I-11 SYNTHESIS AND CHARACTERIZATION OF (CH 3 ) 3 N:BF 3 ................................................... 190 I V I-12 SYNTHESIS AND CHARACTERIZATION OF A CO(III) CAGE COMPLEX .............................. 203 I-13A SYNTHESIS AND CHARACTERIZATION OF A NI(II) MACROCYCLIC COMPLEX .............. 211 I-13B ELECTROCHEMISTRY OF A NI(II) MACROCYCLIC COMPLEX ........................................ 217 I-14 SYNTHESIS AND PURIFICATION OF A PROTECTED PHOSPHINE ......................................... 229 ORGANIC CHEMISTRY EXPERIMENTS .................................................... 243 THE ORGANIC CHEMISTRY LABORATORY................................................................................ 245 LEARNING OBJECTIVES OF THE ORGANIC COURSE ................................................................. 247 O-01: ISOLATION OF PIPERINE FROM PEPPER .......................................................................... 247 O-02: THE BROMINATION OF TRANS-CINNAMIC ACID ............................................................ 252 O-03: SYNTHESIS OF METHYL TRANS-CINNAMATE ................................................................. 255 O-04: REDUCTION OF 3-NITROACETOPHENONE USING TIN AND HYDROCHLORIC ACID ...... 260 O-05: REDUCTION OF 3-NITROACETOPHENONE USING SODIUM BOROHYDRIDE ................... 263 O-06: SYNTHESIS OF N,N-DIETHYL-M-TOLUAMIDE (DEET) ................................................. 265 O-07: SYNTHESIS OF ETHYL 4-AMINOBENZOATE (BENZOCAINE) .......................................... 269 O-08: REAGENT GRADE CHOLESTEROL VIA 5Α,6Β-DIBROMOCHOLESTEROL........................ 273 O-09: THE CONVERSION OF CHOLESTEROL INTO 3β ββ β-CHLORO-5-CHOLESTENE (CHOLESTERYL CHLORIDE)....................................................................................................... 279 O-10: THE ALKYLATION OF Β-DICARBONYL COMPOUNDS ...................................................... 283 O-12: THE SYNTHESIS OF A DIPEPTIDE ..................................................................................... 288 PHYSICAL CHEMISTRY EXPERIMENTS ................................................... 301 PHYSICAL CHEMISTRY REPORT FORMAT ................................................................................. 302 GENERAL HINTS AND INSTRUCTIONS FOR DOING EXPERIMENTS ............................................ 303 THERMODYNAMICS OF NON-IDEAL SYSTEMS ........................................................... 304 P-01 HEATS OF SOLUTION OF POTASSIUM NITRATE ................................................................ 308 P-02 PARTIAL MOLAR VOLUMES IN NACL SOLUTIONS ........................................................... 313 P-04 SOLID-LIQUID PHASE DIAGRAM ....................................................................................... 318 P-05 LIQUID-VAPOR EQUILIBRIUM ........................................................................................... 323 ELECTROCHEMISTRY ........................................................................................................ 327 P-06 CONDUCTANCE OF WEAK AND STRONG ELECTROLYTES ................................................ 331 P-07 ROTATIONAL-VIBRATIONAL SPECTROSCOPY OF HCL .................................................... 336 THERMODYNAMICS AND EQUILIBRIA OF ADSORPTION ON SURFACES ........... 346 P-08 SURFACE TENSION OF BUTANOL SOLUTIONS ................................................................... 349 P-10 LANGMUIR-BLODGETTRY ................................................................................................. 352 P-12 OPTICAL ROTATORY DISPERSION AND CIRCULAR DICHROISM ...................................... 362 P-13 SEDIMENTATION VELOCITY OF BOVINE SERUM ALBUMIN ............................................. 373 P-15 MOLECULAR WEIGHT OF POLYVINYL ALCOHOL BY VISCOSITY .................................... 383 P-16 DETERMINATION OF FORMATION CONSTANTS OF CA-ATP COMPLEXES ...................... 388 P-18 LIGHT SCATTERING ........................................................................................................... 398 P-19 DETERMINATION OF DIFFUSION COEFFICIENT ................................................................ 405 INTEGRATED CHEMISTRY EXPERIMENTS ............................................ 413 INTEGRATED CHEMISTRY LABORATORY EXPERIMENTS ......................................................... 414 X-01 SYNTHESIS AND OPTICAL CHARACTERIZATION OF CDSE QUANTUM DOTS................... 415 X-02 INTRODUCTION TO VACUUM SCIENCE AND MASS SPECTROMETRY ............................... 432 V X-03 SYNTHESIS OF BIS(AMIDATE)BIS(AMIDO) TITANIUM PRECATALYST AND HYDROAMINATION REACTION .................................................................................................. 442 X-04 LASER PHOTOIONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY ............................ 449 X-05 SYNTHESIS AND CHARACTERIZATION OF SWITCHABLE SUPERHYDROPHOBIC- SUPERHYDROPHILIC POLYPYRROLE SURFACES ....................................................................... 462 X-06 INTRODUCTION TO ELECTROCHEMISTRY: VOLTAMMETRY OF FERRICYANIDE ............ 473 X-07 CAPILLARY ELECTROPHORESIS FOR DETERMINATION AND CHARACTERIZATION ....... 486 VI THIRD YEAR CHEMISTRY LABORATORY LEARNING GOALS Students that obtain a passing mark in the third-year integrated chemistry labs (CHEM 315, CHEM 325, CHEM 335 and CHEM 345) will have developed proficiency in the following five broad areas: A. Laboratory Skills A1. Work in a safe manner while in the laboratory. Be able to identify potentially hazardous chemicals and operations before coming to the lab, then act accordingly while doing the experiment. A1.a: General Safety: Obey the safety rules and procedures that are outlined in the lab manual, both general and experiment-specific, at all times. Locate reliable chemical safety information, and from it assess the risks of exposure and the likelihood and severity of exposure. Be able to interpret common hazard symbols (WHMIS and NFPA). Given an experimental procedure, identify the potential chemical and operational hazards posed. Be able to describe the actions that should be taken in the case that a hazardous event occurs. A1.b: Environmental Safety: Dispose of waste properly. Know the common waste streams, which include broken glass, strongly acidic/basic aqueous solutions, aqueous solutions that contain toxic substances, halogenated and non-halogenated organic solvents, and unreactive solids. Be aware that incorrect disposal can lead to unexpected reactions, injuries and/or environmental damage. A1.c: Personal protective equipment (PPE): Wear the appropriate PPE (lab coat, safety glasses) for the duration of the lab period. Wear any additional specialized protective equipment (nitrile gloves, face shield, etc.) as necessary for certain experiments. A2. Perform common laboratory procedures in a proficient and timely manner. Be able to list general procedural steps and/or describe the purpose/use of the procedure or process you are using. A2.a: New skills: Learn new skills as applicable to each experiment you undertake. Be able to follow the instructions provided in the manual to perform these new procedures correctly with little intervention/guidance by the TA. A2.b: Basic skills: Demonstrate a high level of proficiency in common laboratory operations, including (but not limited to) weighing to the correct precision required for the experiment, quantitative transfer of liquids, solids and solutions, preparation of standard solutions, dilutions, heating (hot plate, heating mantle, oil bath), and measuring temperature. A2.c: Discipline-specific skills: Proficiently perform the skills taught in second-year lab courses, such as recrystallization, reflux, distillation, titration and measurements of volume, temperature and pressure. Be able to describe the applications and limitations of a range of instruments and be able to select appropriate instrument(s) to satisfy a specific scientific need. VI I A2.d: Clean-up: Clean all glassware and work areas (around sinks, balances, benches and instrumentation) before you leave the lab. Report deficiencies (missing items, empty containers, broken equipment etc.) to the “in-charge” person immediately. A3. Maintain an up-to-date, detailed, research-grade laboratory notebook. Use a laboratory notebook that has sequentially numbered, duplicate pages. Record information in either blue or black ballpoint ink. Title and date every page. Maintain an up-to-date table of contents. Submit the top copy of each page with your report; keep the copy page in your notebook to retain a complete record of your experimental work. Neatly cross out errors (never use correction fluid or tape). Cross through, but do not remove, any blank pages left between experiments. A3.a: Document laboratory procedures: Document all procedures, observations and data collection at the time they occur, with sufficient detail such that a competent scientist who is unfamiliar with the work can reproduce the experiment. A3.b: Accurately record data and observations in an organized way: Plan for and prepare data tables (as needed) for each experiment. Record both qualitative and quantitative observations. Organize electronic files logically and with descriptive file names. Record numerical data to the number of significant digits consistent with the measuring device used. Include units consistent with the conventions of the measurement and discipline. A4. Apply appropriate methods of analysis to data and present raw data using meaningful units. Select the appropriate analysis approach for an experimental procedure, including the choice of statistical analysis applied to the data and the presentation of the results in a graphical format following the conventions of the discipline. A5. Perform logical troubleshooting of laboratory procedures and instrumentation. Based on experience in prior lab courses, anticipate potential problems and devise strategies that can be used to overcome any problems. Use logical process(es) of elimination to narrow down the source(s) of the problem. Attempt to solve the problem yourself, but also know when to ask for help. Using appropriate terminology explain the problem and describe the steps you took to solve it. B. Written and Oral Communication B1. Report your experiment and its results in a clear and concise manner. Accurately summarize the principles underlying the experiment in your own words. Produce schematics to describe experimental set-ups and processes. Present data clearly in tables and graphs. Use units, errors and significant figures correctly. VI I I B2. Write in scientific style and with appropriate depth. Write clear and well-organized laboratory reports. Discuss results in detail and explain why they agree/disagree with theory. Write compelling scientific arguments in support of your conclusions. B3. Access and properly cite scientific literature. Cite scientific literature following the conventions of peer-reviewed scientific journals; cite within text and with an appropriate reference list. Find and cite relevant textbooks, journal articles and websites. B4. Build competence in scientific dialogue. Use correct terminology. Demonstrate an appropriate level of confidence in verbal explanation of scientific reasoning. C. Responsibility and Professionalism C1. Effectively prepare in advance for laboratory work. Perform reading, calculations and background research as needed to maximize learning during in-lab time. Be able to explain the reasoning behind every step in the experiment. Develop your own questions to be answered during the lab. C2. Learn from mistakes. Use feedback from the teaching staff to avoid repeating mistakes. Anticipate problems and challenges. Be alert to unexpected results, hypothesize the source(s), and resolve as time permits. C3. Work as a team. Effectively and efficiently work with a partner during in-lab time. (Note that some experiments require only individual, not group work). Establish responsibilities and/or roles; monitor progress and adjust as needed to successfully complete the experiment. Collaborate and communicate effectively with your lab partner. C4. Demonstrate high ethical standards. Demonstrate high ethical standards in data collection, lab report writing and personal behaviour. Recognize the point at which work should diverge from being shared with your partner to being an individual effort. Distinguish healthy discussion and team work from plagiarism. Be aware that report writing is an individual activity. Understand the basic principles of intellectual property. D. Context This section summarizes some of the broader goals of the CHEM 3XX third-year integrated chemistry labs. I X D1. Become proficient in a range of modern techniques. Become proficient in the use of a wide range of modern techniques relevant to chemical research. Describe the capabilities and limitations of the techniques you use. D2. Develop awareness of the interdependence of the traditional sub-disciplines of chemistry. Chemical research and scholarship has traditionally fallen into a number of sub-disciplines, including analytical, inorganic, organic and physical chemistry. The boundaries between these sub-disciplines are however becoming less clear and future chemical innovation will require an understanding of the potential contributions of each of the sub-disciplines to solve a given problem. In this course you are encouraged to make links between the sub-disciplines by, for example, carrying out one or more of the “integrated” experiments where appropriate. D3. Become comfortable in an interdisciplinary research environment. Reaching even further across disciplinary boundaries, important discoveries are increasingly made at the interfaces between chemistry and other fields. This course aims to prepare you for this “interdisciplinary future”, for example by encouraging you to appreciate that procedures are driven by scientific need and not by available equipment or disciplinary traditions. E. Integration and Application of Knowledge/Experience E1. Apply critical thinking in the laboratory. Make strategic decisions by thinking critically about observations made in the lab. Use these observations to identify possible options, evaluate choices, and implement and justify course(s) of action. E2. Recognize whether results and conclusions "make sense". Interpret various types of data to draw defendable and relevant conclusions. Continue to develop your ability to recognize when data, conclusions, and results from calculations “make sense”; estimate magnitudes of quantities and check units resulting from calculations. X Laboratory Director Contact Information Analytical Dr. Robin Stoodley Office: C226B Email:
[email protected] Tel: (604) 827-5829 Inorganic Dr. Vishakha Monga Office: B470A E-mail:
[email protected] Tel: (604) 822-3678 Organic Dr. Christine Rogers Office: C326C E-mail:
[email protected] Tel: (604) 827-4820 Physical Dr. Guillaume Bussiere Office: B450A E-mail:
[email protected] Tel: (604) 822-6384 Where to find us: XI XI I Your Personal Laboratory Schedule September – December 2013 Date Started Expt. Code TA Name TA Signature **If you are taking C315 you will only use half of these spaces. You will fill in all of these spaces if you are taking C325.** XI I I Your Personal Laboratory Schedule January – April 2014 Date Started Expt. Code TA Name TA Signature **If you are taking C325 you will only use half of these spaces. You will fill in all of these spaces if you are taking C345.** XI V Laboratory Logistics Check-In week **Read through the introductory material in this lab manual. Pages IX – XVI. **Arrive to your laboratory orientation session. Students who are not present during their orientation will have their registration terminated. According to the Faculty of Science Academic regulations: “Students registered in any Science course that has a laboratory must attend their first scheduled laboratory class in that course. Failure to do so will result in the termination of student’s registration in the course. Students who are unable to attend their first scheduled laboratory class in a course must notify the head or the designate 1 , of the department concerned within 48 hours of the time affixed for that class or have their registration in the course terminated.” How the labs are run Multiple experiments run simultaneously in each lab period; students cycle through a subset of these experiments over the course of ten weeks. The labs are to be completed in four hours. TAs are present; each takes care of a set of experiments. Some labs are performed in pairs, however, each student must submit their own unique lab report. Safe laboratory practices are essential. Eye protection and labcoats must be worn. Keep all instrumentation clean - especially the analytical balances. Keep the laboratory bench clean. Your TAs will inspect your work area before you leave the laboratory. Missing a laboratory session The CHEM 315/325/335/345 lab is running above capacity – Make-up labs can only be scheduled with difficulty. Make-up labs may be scheduled outside your normal lab period or at the discretion of the lab directors. Standard university policy for academic concessions applies. Labs missed due to illness can be re-scheduled; a doctor’s note must be provided. Contact the specific lab director in advance if you know you will miss a laboratory session. If you miss a lab due to illness, you must hand-in any lab report that was due in the missed session as soon as you return to attending classes. Do not wait until your next lab session in the following week! If you are late arriving to the lab, a penalty of 10% may be applied to your report mark; depending on circumstances you may have to forfeit the lab with a grade of zero. XV Course Designates: Dr. Robin Stoodley [Analytical]; Dr. Vishakha Monga [Inorganic]; Dr. Christine Rogers [Organic]; Dr. Guillaume Bussiere [Physical]. XVI During The Lab Session These experiments are designed to fit into the four-hour time allotted. However, you must be familiar with the experiment and know what you are to do before you arrive in the laboratory. Additionally, you are responsible for reading the preliminary pages of each discipline; Analytical, Inorganic, Organic, and Physical. The most important pre-experiment preparation is to read the manual! WEAR YOUR LAB COAT AND EYE PROTECTION. No open-toed shoes allowed in the lab. Tie long hair up or back – you don’t want it falling into your beakers! Be respectful of everyone in the lab. Put equipment back into the appropriately-labeled lockers and return unknown sample vials back to the dispensary. You are not the only one performing each experiment and no one wants to clean up after you. There is one accepted lab notebook for CHEM 315/325/335/345. It is the Hayden McNeil spiral bound Student Laboratory Notebook. This can be purchased from the UBC bookstore. UBC disability policy: http://www.universitycounsel.ubc.ca/policies/policy73.html Anyone who has a disability that requires special testing accommodation or other class modifications should bring the request for accommodations or for changes in the accommodation needs to the attention of appropriate personnel in a timely manner in order to allow for arrangement of accommodations. All new and returning students who will be requesting an accommodation are required to contact the Disability Resource Centre (DRC) at the beginning of each term. At the beginning of each term, all students should discuss their situations with each instructor from whom they are seeking accommodation. DRC will contact instructors prior to this meeting if requested to do so by the student. Contact the DRC at Brock Hall 1203, 1874 East Mall, Telephone 604-822-5844. XVI I Lab Reports There is a general grading structure for lab reports submitted in CHEM 315/325/335/345. Please see the specific report format required for each discipline. Each 4 hours lab period corresponds with a report that is marked out of 20. This means: *CHEM 315/335 9 (4 hour lab periods) x 20 180 marks total *CHEM 325/345 18 (4 hour lab periods) x 20 360 marks total *Students registered in CHEM 325 or 345 MUST separately obtain a passing grade in each discipline (analytical, inorganic, organic, physical, and -if selected as part of your course schedule- integrated). *Combined Major in Science students registered in CHEM 315 or 335 MUST separately pass each discipline that appears in your schedule of selected experiments. *Biochemistry students registered in CHEM 315 or 335 MUST separately pass each of organic and physical. XVI I I Academic Honesty Students are expected to follow the University of British Columbia academic integrity guidelines, which can be found at the website: http://www.calendar.ubc.ca/vancouver/index.cfm?tree=3,54,111,959 Please read the statement reproduced below, and note particularly the section italicized here for emphasis, but not in the original document. Upon registering for courses, you have entered into a contract with the university agreeing to the statement below. “Plagiarism, which is intellectual theft, occurs where an individual submits or presents the oral or written work of another person as his or her own. Scholarship quite properly rests upon examining and referring to the thoughts and writings of others. However, when another person's words (i.e. phrases, sentences, or paragraphs), ideas, or entire works are used, the author must be acknowledged in the text, in footnotes, in endnotes, or in another accepted form of academic citation. Where direct quotations are made, they must be clearly delineated (for example, within quotation marks or separately indented). Failure to provide proper attribution is plagiarism because it represents someone else's work as one's own. Plagiarism should not occur in submitted drafts or final works. A student who seeks assistance from a tutor or other scholastic aids must ensure that the work submitted is the student's own. Students are responsible for ensuring that any work submitted does not constitute plagiarism. Students who are in any doubt as to what constitutes plagiarism should consult their instructor before handing in any assignments.” If you include in your laboratory reports any phrases, sentences, or other material of which you are not the author, they must be placed in quotations and cited/referenced. Figures, graphs, etc. of which you are not the author must be cited and referenced. There is a zero-tolerance policy for academic dishonesty in the CHEM 315/325/335/345 laboratory. Violations of these guidelines may result in academic discipline ranging from a zero mark for a laboratory report up to expulsion from the university. Remember, however, that the inclusion of material written by others often does not demonstrate to the marker your understanding of the subject. As such, simply quoting others’ work is insufficient to earn full marks. Normally, you will submit a hardcopy (i.e. paper copy) of your lab reports. However, you may be asked to submit an electronic copy. Electronic copies will be submitted to a service to which UBC subscribes, called Turnitin (www.turnitin.com). This is a service that checks textual material for originality. Failure to submit an electronic copy will minimally result in a zero grade for that assignment. Please see your instructor for further information. XI X Safety Instructions To avoid injury to yourself and others, you are required to follow the safety rules below. Failure to comply with these rules while in the laboratory may result in suspension or expulsion from the course. 1. If you have a medical problem or condition that may affect your performance or safety in the laboratory, you must discuss it in private with the laboratory coordinator. This information will be held in strict confidence. 2. EYE PROTECTION a) Adequate eye protection is required for all individuals working in the laboratory. Do not remove your eye protection until you have physically left the lab room. The following types of eye protection are acceptable: b) Normal prescription eyeglasses or safety glasses, either with or without safety side- shields, as long as the lenses are shatterproof (i.e. plastic) and cover a large enough area surrounding your eye (this usually means that the frames must be a minimum of 4 cm from top to bottom as well as from side to side). c) Safety goggles that form a tight seal to your face. For some labs (particularly organic), where exposure to toxic or irritating fumes is a real problem, the best form of eye protection is safety goggles. 3. Eye injuries, whether chemical or mechanical, must always be considered serious. IN CASE OF CHEMICAL INJURY TO THE EYE THE BEST PROCEDURE IS IMMEDIATE PROLONGED CONTINUOUS FLUSHING WITH WATER (15 - 20 minutes) at an eyewash fountain. Eyes must be forced open to be washed well. 4. ALL-COTTON LABCOATS MUST BE WORN WHEN WORKING IN THE LABORATORY. 5. FLUSH WITH WATER ANY PART OF YOUR BODY THAT COMES IN CONTACT WITH ANY CHEMICAL. Use lots of water. Removal of clothing may be required. 6. Do not wear open-toed shoes or sandals. Tie-up long hair. 7. NEVER EAT OR DRINK IN THE LABORATORY. 8. Throw away cracked or chipped glassware immediately into the glass waste container. 9. Clean up chemical spills immediately. Check with the instructor for the proper procedure. Ask your instructor about the disposal of used chemicals. Waste containers for disposal of halogenated organic waste, non-halogenated organic waste and radioactive waste. 10. WASH YOUR HANDS WHEN LAB WORK IS FINISHED. NOTE: The wearing of contact lenses in the chemical laboratory has historically been perceived as a safety hazard. The concerns are: a) if chemicals are splashed into the eyes, they can become trapped under or absorbed by the lenses; b) some chemicals can cause coagulation of the protein in the eye within seconds; c) chemical vapours can be trapped under or dissolve into soft lenses. It is also extremely difficult to remove lenses if something has been splashed into your eyes. More recently, both the American Chemical Society and the (American) Occupational Health and Safety Administration have issued statements indicating their studies suggest the wearing of contact lenses does not pose any additional hazard and can be treated as safe if and only if safety glasses or goggles are also worn. XX Safety Education/Training Program WHMIS ("Workplace Hazardous Materials Information System") training is required to ensure that workers are able to apply their safety knowledge to protect their health and safety. Instruction on the content and significance of labels and MSDS, emergency procedures, and information and procedures for safe use, handling, storage and disposal of hazardous materials are all part of the training course. Some products are exempted from federal WHMIS requirements for labels and MSDS if they are already covered by other labeling legislation. These products include: cosmetics and drugs explosives pesticides radioactive substances some consumer products Certain products are completely exempted from both federal and provincial WHMIS requirements. The following are included in this category. wood and wood products manufactured articles tobacco and tobacco products goods handled, offered for transport or transported pursuant to the Transportation of Dangerous Goods Act. Important note: WHMIS training is available to all UBC students free of charge. If you would like to register for this training visit the following URL. http://www.hse.ubc.ca/crs_reg/start.asp The laboratory safety training courses are offered several times a year. It is recommended that you enroll in this course. Safe Handling of Materials - WHMIS Labels and the MSDS Before using any chemical you must be aware of any hazards involved in its handling, and the precautions which should be taken. All controlled product containers have a WHMIS label which is a good primary source of information. The symbols used on these labels are described below. "Material Safety Data Sheets" (MSDS) provide detailed and comprehensive information on material hazards. A website with a database of MSDS is: http://ccinfoweb.ccohs.ca/msds/search.html XXI Working Safely in Chemistry Laboratories It is inevitable that you will be required to handle hazardous substances outside of the fume hoods, given the current facilities available. The following information has been provided in the event that you have concerns about safety practices in the undergraduate chemistry laboratories. The pre-lab talk, whether presented by a lab director or a T.A., will contain important safety information for that laboratory period - take note! Topics such as personal protective equipment needed (in addition to safety glasses and lab coat), the generation of corrosive gaseous by- products that require a gas trap, safe heating of flammable liquids etc. will be highlighted. The material presented during the pre-lab talk will, in part, reinforce the safety information readings that you are required to do prior to coming to the lab. Make note of the physical properties of the chemicals that you will be handling and generating, as well as specific hazards and remedies. Material Safety Data Sheets (MSDS) are readily available online from the chemical suppliers listed below: Sigma-Aldrich http://www.sigmaaldrich.com/canada-english.html Fisher Scientific http://iris.fishersci.ca/MSDS2.nsf/Search?OpenForm TCI America http://www.tciamerica.com/ Alfa Aesar http://www.alfa.com/en/gh100w.pgm Acros Organics http://www.acros.com/Welcome.aspx Praxair http://www.praxair.com/na/ca/en/can.nsf Strem http://www.strem.com/ The UBC Department of Chemistry web page also has Health and Safety links (http://www.chem.ubc.ca/health-and-safety). If you have safety questions during an experiment, speak with your T.A. and/or the lab director. If the answers provided do not satisfy your concerns, contact the Chemistry Department Safety Officer (604-827-5216, Room A237). Be prepared to provide specific information about the circumstances, such as the date, the chemicals being used, and your particular concerns. UBC Risk Management Services (604-822-2029) is the final level for dealing with safety issues on campus. XXI I Emergency and Non-Emergency Phone Numbers EMERGENCY NUMBERS From cell phones From office phones Emergency Fire, Police, Ambulance, Hazardous materials response 911 9-911 UBC Emergency / First Aid 604-822-4444 2-4444 Campus Security 604-822-2222 2-2222 UBC Hospital Urgent Care Department 604-822-7222 2-7222 NON-EMERGENCY NUMBERS From cell phones From office phones Ambulance 604-872-5151 9-604-872-5151 Campus Fire Department 604-665-6010 9-604-665-6010 Student Health Services 604-822-7011 2-7011 Chemistry Dept Safety Office 604-827-5216 7-5216 Risk Management Services 604-822-2029 2-2029 XXI I I Hazard Symbols and Classes 24 ANALYTICAL CHEMISTRY EXPERIMENTS Did you know? Payscale.com reports Canadian analytical chemists salaries based on national data. In July 2013, the salary range for an analytical chemist with two years experience was approximately: $35,240 (10th percentile) to $69,055 (90th percentile). Vancouver has the lowest average pay compared to other major Canadian cities. 25 Analytical Chemistry Laboratory Course The Analytical Chemistry laboratory course is designed to introduce you to instrumental analysis, to improve your technical lab skills and to provide you opportunities to improve your report-writing skills. The instrumentation used in this course is complex; in addition to working with the data that is output, you will be expect to become familiar with all aspects of the instruments’ design and operation. Analytical chemistry methods such as use of calibration curves, standard addition and internal standard must be mastered. Learning Objectives of the Course The goal of the Analytical Chemistry laboratories is to equip you with skills and abilities that you will need as a practicing scientist. These include being able to design experiments, safely undertake them, and interpret their results. Listed below are learning goals 1 to attain in the Analytical Chemistry laboratories. The goals have been classified by type. Safety: Identify and evaluate health, safety and environmental risks associated with your lab work. Use best practices to minimize risks. Teamwork: Effectively and efficiently work with a partner during in-lab time. Determine responsibilities and/or roles; evaluate progress and adjust accordingly to finish on time. Models: Evaluate advantages and disadvantages of different theoretical models to understand their success at mimicking experimentally-observed behaviour. Apply your own experimental data in support of a known model or evaluate whether or not a model sufficiently explains your experimental data. Experimentation: Determine a procedure including selection of instrumentation, methods, and approaches; conduct the experiment; collect data. To an appropriate level, demonstrate and apply trouble-shooting or problem-solving techniques. Instrumentation: Make measurements of concentration or amount using appropriate instrumentation, equipment, and software. Hands-on: Demonstrate proficiency in hands-on lab skills such as pipetting, filling of volumetric flasks, injection techniques, etc. Maintain a clear and comprehensive lab notebook. Analyze: Improve your ability to evaluate, interpret and analyze your data. Apply calculations to your raw data as needed; respect unit conventions and show judgment with respect to magnitude of result. Using the data, draw conclusions and support them. Learning from failure: Back-rationalize possible causes of unusual or incorrect data. Identify possible solutions; implement them as time permits. Communication: Using the format of a formal lab report, communicate clearly and concisely the context, the background, your data, your results and the conclusions of your experiment. Ethics: Demonstrate high ethical standards, including in data collection, writing of lab reports, and personal behavior towards students, staff and faculty. 1 Learning goals adapted from Feisel, L.D.; Rosa, A.J. The Role of the Laboratory in Undergraduate Engineering Education. J. Engineering Education 2005, 94, 121-130. 26 Analytical Grading Structure Each experiment is marked out of 20. The general breakdown is: Technique: 2 marks for working carefully and competently, and in cleaning up. Calculations: 2 to 4 marks for numerical treatment of data. Take care to avoid errors due to incorrect or missing dilution factors. Data: 7 marks based upon a comparison of your result with that expected for the unknown. Note: 1 mark deducted if your result is not given on the 1 st page of your report; 1 mark deducted if the result has missing or incorrect units; 1 mark deducted for missing or incorrect unknown number. In case of calculation error(s) that impact your reported result, you may correct your mistake and resubmit a new result. This can be done once per experiment. Guessing is not tolerated. Report: 7 to 9 marks based upon the report as outlined in the manual. Late Submissions 2 marks will be deducted for each week, or part thereof, that your report is late. The absolute final deadline for submissions is 4:00pm Friday November 29 th 2013 (Term one) and 4:00pm Friday April 8 th 2014 (Term two). No consideration will be given for reports that are lost or submitted late as a result of computer or printer failure. Save your work frequently! Handwritten reports are acceptable but are strongly discouraged. Mark Normalization Please note that the lab marks assigned by your TA may be subject to normalization. If your TA is an unusually generous marker, the grades will be normalized downwards. If your TA is an unusually difficult marker, the grades will be normalized up. Usually, little normalization is required. Notebook Guidelines and Evaluation You will use your lab notebook to prepare for each laboratory session ahead of time and during the lab itself. All calculations and discussion etc. will be handed in as a separate report to your TA. The following are some guidelines about laboratory notebooks: Your notebook must be in the laboratory when you are. It must be a bound notebook with numbered pages and a table of contents. Leave space for the table of contents at the beginning of the book and update as necessary. Entries must be made in ink, and there must be no erasures or white-outs. Draw a single line through entries that are incorrect or otherwise not needed. Numerical data must have units included. Do not remove pages from your lab notebook under any circumstances. It is a good idea to keep the left-hand pages blank for later additions or corrections or comments. 27 A very good article on laboratory notebooks is "Keeping a Laboratory Notebook" by Eisenberg, A. Journal of Chemical Education 1982, 59, 1045-6. To prepare for the lab, each notebook entry should contain: Title of Experiment and Date Purpose of Experiment (1-2 sentences describing the purpose- make sure this is correct!) A brief description of what you will do in the experiment and how it will be done (~3- 5 paragraphs) Calculations related to solution preparation Laboratory Reports Full lab reports are required for all experiments. Your report should include sections of Introduction, Instrumentation/Method, Data, Calculations, Results and Discussion. Reports are normally due one week after the experiment was performed. Where the schedule is interrupted (e.g. by reading break) the report must be submitted at the next scheduled lab period. Each student must prepare his or her own report independently, even when the laboratory experiment is carried out in pairs. Reports must be submitted directly to your teaching assistant. A penalty will be assessed for late reports. About the Unknowns There are two methods used to prepare the unknowns. Depending on which method is used, your first step in the experiment is either to dilute the entire provided unknown into a volumetric flask or to pipette 10.00mL from the unknown vial into a volumetric flask. It is very important that you do the correct one: the lab manual will give you the correct procedure for each experiment. In both cases, when calculating the concentration of the unknown the volume provided can be taken as 10.00 mL. The value(s) given on the front page of your report must be the concentration of the unknown in the original vial. 28 Preparation For and During the Lab Write your report’s “Principle of Method” section before coming to the laboratory. This is intended as lab preparation, and 1 technique mark will be deducted if it has not been done. You may revise this section after the experiment to reflect new understanding or insight gained during the experiment. Your pre-lab preparation should also include the procedure you will follow for preparation of standard solutions (i.e. dilution calculations). Do some preliminary calculations and graphing on your data before you clean up and leave the laboratory. The time to find out whether your data is satisfactory is while you are in the laboratory and are able to repeat a questionable measurement. Don’t dispose of your solutions until you have done this! Unexpected or unusual results should be brought to the attention of the instructor or the TA as soon as possible. Cleanliness is of great importance in the analytical lab. Carefully clean all glassware with tap water, then rinse with deionized water using the squeeze bottles provided. A soap solution for cleaning is available on the bench. Note experiment 11 is extremely sensitive to contamination: separate instructions for acid-cleaning the plasticware used for trace-level copper analysis are provided. 29 Analytical Chemistry Report Format COVER PAGE: Student name, course number and section, name of partner (if applicable), date of experiment, unknown number, the result for the concentration of your unknown (as supplied in the vial) with the uncertainty and associated confidence level. All results should be reported in units of g/mL. TITLE: This should give the nature of the analytical method and of the analyte. Please avoid simply copying the title used in the lab manual. ABSTRACT: A brief (3-4 sentences; not more than 8 lines) description of the experiment in your own words. Include what you did in the lab, how you did it, and what the results were. PRINCIPLE OF METHOD: The principle of method section should be written before you attend the laboratory. 1 technique mark will be deducted if you have not completed it before the lab period or if it is incomplete. Once you have gained a better understanding of the experiment by doing it, you should improve your principle of method by including your new insights. Describe briefly the chemical and physical principles relevant to the experiment. Use diagrams, graphs, or chemical equations where appropriate. Include any relevant laws or equations. Describe the experimental set-up (instruments) and use a schematic diagram where applicable. Explain the functioning of the main components of the instrument. Do not describe details of procedure. Your goal in this section should be to convince the TA that you fully understand the methodology used in the experiment. RESULTS AND CALCULATIONS: A worked-example of each type of calculation should be given. Data should be presented in tables with a table number, a title and units. Each graph should have a graph number and a title with labels on both axes. Every graph should be on its own page and should fill that page. Graphs and tables should be used to help the reader understand the results; they should always be referred to in the text of the report. Rescale graph axes to illustrate the points you wish the reader to understand. For example, if the intercept of a line is used in your calculations, your graph should be formatted to show the intercept. Do not put raw data into this section; use an appendix instead. DISCUSSION: This is the most important part of your report. This section is the place where you tell the reader what the experiment was all about and what it has shown. Examine the analytical method critically, based on your knowledge and experience. Discuss your results specifically. Do they agree with theory? Describe the use of any special analytical techniques (spiking, standard addition, etc.?) How did these techniques affect your results? Comment on any spectra or calibration plots used. Comment on their meaning, linearity, etc. How do they relate to quantitation of the analyte? Discuss the numerical result(s) obtained. If you used more than one instrumental method or data analysis treatment compare them and the results they produced. Consider both precision and accuracy. 30 ERROR LIMITS: Give an estimate of the error limits for all reported numerical quantities (see discussion of error analysis in your textbook). List sources of systematic error and, if possible, estimate their limits. Estimate random error limits from the observed scatter of data. This may be done by statistical computation (regression analysis), or graphically in the case of gross error. Try to determine which of random or systematic errors predominates. Don’t focus solely of uncertainties in glassware volumes unless these are the main sources of error. Describe what can be done to minimize errors. This section may be combined with the discussion if desired. CONCLUSION: State your major findings in a few lines. REFERENCES: Cite your sources: manuals, journal articles, textbooks, websites, etc. in ACS format. (See policy on plagiarism) APPENDIX (if applicable): An appendix is used to include material which is not properly formatted: raw data, details of calculations and error propagation. If you choose to include an appendix, you must refer to it in the body of your lab report. Improved clarity is the main reason for including an appendix. Consider the TA who has to read your report; all else equal, unclear lab reports receive lower grades. As a rule of thumb for lab reports, remember that your report is a written communication from you to show your TAs all that you know. Part of your mark will come from how well you communicate your knowledge to your TA. 31 Accounting for Uncertainty I. AN ERROR IS AN ERROR IS AN ERROR. We never know exactly how big an uncertainty should be associated with a measurement; we even don’t know exactly the range in which a quantity we are trying to measure lies. Much error analysis consists of educated guesses. Statistical analysis can help, but to use them properly we must understand that they do not remove uncertainty, but merely let us express more clearly what the uncertainty is. For example, the statement: [Mn 2+ ] = (1.56 "0.03) H 10 -4 g/ml does not mean that [Mn 2+ ] lies, for sure, between 1.53 and 1.59 H 10 -4 g/ml. It may mean several things, according to the convention used by the particular writer for the meaning of 0.03. To avoid ambiguity, state your convention along with the numerical result, for example, [Mn 2+ ] = (1.56 " 0.03) H 10 -4 g/ml (95% confidence limits). This means: "There are 19 chances out of 20 that [Mn 2+ ] lies between 1.53 and 1.59 H 10 -4 g/ml." II. SYSTEMATIC AND RANDOM ERRORS 10 ml of a solution of Mn 2+ was diluted to 100 ml in a volumetric flask, and the manganese in the diluted solution was determined by some analytical method. In several repeats of the same experiment, results were: 1.54, 1.58, 1.53, 1.55, 1.60 ... H 10 -5 g/ml. Mean [Mn 2+ ] (diluted solution) = 1.56 H 10 -5 g/ml. Mean [Mn 2+ ] (before dilution) = (1.56 H 10 -5 ) H (100/10) g/ml = 1.56 H 10 -4 g/ml. RANDOM error is usually estimated from statistical analysis of the scatter in results of repeat tests, such as the set of five given above. SYSTEMATIC error can arise in many ways, some of which may not come to mind very easily. For example, the dilution factor 100/10 in the above calculation is very likely to conceal systematic error. Commonly, we would use the same volumetric flask in every repeat of the same experiment, and its volume might not be exactly 100 ml. Suppose, for simplicity, that the 10 ml sample volume were exact, but that the flask for dilution actually had a volume of 102 ml (a great exaggeration of the likely error in a volumetric flask, just for the sake of illustration). Then: Mean [Mn 2+ ] (before dilution) = 1.56 H 10 -5 H (102/10) = 1.59 H 10 -4 g/ml. The previous estimate contained a systematic error of -0.03 H 10 -4 g/ml. Note that a systematic error, being always one way, has a sign, while random error consists of scatter both positive and negative, and is expressed as (quantity) " (random error). We cope with systematic error by using common sense and scientific experience to list the likely sources of it and devise procedures to eliminate as many as possible. For what sources remain, we have to make educated guesses. A large part of error analysis necessarily consists of these guesses. 32 Suppose, for example, that one had a couple of dozen volumetric flasks, and took a different one off the shelf for each repeat of the experiment. Is the error in the volume of the flask now systematic, or random? This depends on what was going on in the factory where the flasks were made. The manufacturer may or may not have done something which made all the flasks consistently too big. One can only guess about this kind of thing unless one has much information about how equipment was made and calibrated, right back to the primary standards of mass, length, time, temperature, etc. Fortunately, when experiments are being done to about three-figure accuracy, the expected range of error in calibrations of volumetric equipment or of the weights built into analytical balances is usually very small in comparison to other sources of error (table 1). The purpose of the above example was not to get you locked in to worrying about volumes, but to encourage you to be wide-ranging in your thoughts about what could have gone wrong in your attempt to measure something quantitatively. A large part of an error analysis should consist of your assessment, in words, of the most likely sources of large error. TABLE 1: Reasonable Error Limits Titre by buret (50 ml) 0.03 ml End point detection 0.03 ml Volumetric flask (50 ml) 0.05 ml " (100 ml) 0.08 ml (250 ml) 0.12 ml Pipetting (5 ml) 0.01 ml Allow 15s drain time “ (10 ml) 0.02 ml Allow 15s " (25 ml) 0.03 ml Allow 25s Weighing by analytical balance 0.0001g III. STATISTICAL ANALYSIS OF RANDOM ERRORS If you set out to measure the numerical value of some quantity, you start with the assumption that there is a "true value" to be found. You can never determine exactly what it is, but you can try to obtain better and better approximations, if you have enough time available. The ways to reduce errors are quite different for systematic and random errors. For systematic errors, you have to think what they may be and change your procedures so as to eliminate them as much as possible. For random errors, you can reduce them by repeating the same experiment many times and averaging the results. The mean value x of the quantity should be an increasingly close approximation to the "true value" as the number of determinations (n) increases. But the mean becomes exact only if n is infinite. Unfortunately, the error limits decrease with the square root of n, so that it takes an increase of a factor of 4 in number of experiments to decrease error limits by a factor of 2. These ideas are expressed statistically in the quantities known as variance, standard deviation, standard deviation of the mean, and confidence limits. Variance is simply the square of the standard deviation F, and one usually needs to deal only in terms of the latter. F is a characteristic of the scatter about the mean value. It should not get smaller as n increases, but, once you have done a fairly large number of determinations, should remain roughly constant however many more you do. Standard deviation is calculated for a set of determinations x 1 , x 2 ...x i ...x n of the same quantity by: F 2 = (x i - x ) 2 /(n - 1) i n = ∑ 1 33 The precision, or reproducibility, of a measured value, is related to the standard deviation. Once you have found a standard deviation, by the above procedure, it can in fact be used as a measure of precision for any one determination in the series of n. But since you have made n determinations, you can get better precision by averaging the whole set. The precision of the mean value is given by a quantity known as the "standard deviation of the mean" which is: F m = F/n 1/2 Since F is roughly independent of n, F m decreases as n increases; that is, one can improve precision in the presence of random errors simply by doing the experiment more times. But it takes four more times to halve the error limits. These limits are quoted in different ways by different people. Some tend to use "F m as the error limits. These are 68% confidence limits, i.e. there are about 2 chances out of 3 that the true value lies within the limits stated. This isn't a remarkably good chance, and many people quote wider limits. Roughly, "2F m gives 95% confidence limits, i.e. 19 chances out of 20 that the true value lies within the limits. This 95% confidence limit is widely used in analytical chemistry. ACCURACY, PRECISION, ACCEPTED VALUE, TRUE VALUE One never knows the true value of any quantity; but for many quantities there is an "accepted value", being the result obtained in the experiment which is generally judged to have been the best performed to determine this quantity. ACCURACY of any determination means the closeness with which the determination matches the accepted value, or, the closeness you think it has to the true value. This must again be expressed in terms of "19 chances out of 20 … ", etc., but it includes the effects of both random and systematic errors. PRECISION, as discussed above, relates only to random errors. IV. QUANTITIES CALCULATED OUT OF SEVERAL MEASURED QUANTITIES, Z = f(A, B, C). Suppose that we know the error limits (e.g. 95% confidence) for A, B and C. What are the error limits of Z? Strictly, it depends whether the errors are systematic or random, according to Table 2. In practice, we often don't know precisely what the mix of random and systematic errors (think, once again, about those volumetric flasks and their manufacture). Hence, it is common to use the method of calculation shown in Table 3, which isn't the proper way to do it for either kind of error, but is the best we can do for an unknown set of errors. For random errors, )A, etc., in these tables is the quantity you are putting after ", i.e. 68% or 95% confidence limits. For systematic errors, )A has, in principle, a definite sign, but if you know what it is the error has ceased to be an error. 34 TABLE 2: Propagation of error for systematic errors and for random errors Computation Systematic errors Random errors Addition or Subtraction Z = A + B - C C B A Z ∆ − ∆ + ∆ = ∆ 2 2 2 2 C B A Z ∆ + ∆ + ∆ = ∆ Multiplication or Division Z =AB/C C C B B A A Z Z ∆ − ∆ + ∆ = ∆ 2 2 2 2 C C B B A A Z Z | ¹ | \ | ∆ + | ¹ | \ | ∆ + | ¹ | \ | ∆ = | ¹ | \ | ∆ General Z=ƒ(A,B,C) C C B B A A Z ∆ ∂ ∂ + ∆ ∂ ∂ + ∆ ∂ ∂ = ∆ f f f 2 2 2 2 2 2 2 C C B B A A Z ∆ ∂ ∂ + ∆ ∂ ∂ + ∆ ∂ ∂ = ∆ | | ¹ | \ | | | ¹ | \ | | | ¹ | \ | f f f TABLE 3: Propagation of error for an unknown mixture of systematic and random errors Computation Errors Addition or Subtraction Z = A + B - C C B A Z ∆ + ∆ + ∆ = ∆ Multiplication or Division Z =AB/C C C B B A A Z Z ∆ + ∆ + ∆ = ∆ General Z=f(A,B,C) C C B B A A Z ∆ ∂ ∂ + ∆ ∂ ∂ + ∆ ∂ ∂ = ∆ f f f V. SLOPES OF STRAIGHT LINES When a graph of y versus x should be a straight line, it is common to calculate its slope m and intercept b in the equation y = mx + b statistically, by the method of least squares. This is a good procedure, but it has some pitfalls which should always be borne in mind: 1. Always draw the graph, for two reasons: (a) Plotting the least squares line on the graph is a good check against numerical error in putting data into the calculator. The line should clearly be a very good fit to the points. (b) The words "should be a straight line" in the first sentence above don't really mean very much. You may find that the plotted points clearly fit a curve, and that you shouldn't be looking for a straight line. 2. In some experiments, only part of the data fit a straight line, and there is curvature in some other region. In this case, you make a visual choice from your graph of which points to take into account in drawing the line. There is then no point in using anything other than a visual procedure for drawing the best line. 3. The usual method of doing the least squares calculation assumes no error in x, and random scatter in y. For example, if you are constructing a calibration curve of spectroscopic 35 absorbance against concentration of a coloured solute, the absorbance commonly may have errors amounting to 1%, while the standard solutions can be made up to an accuracy of 0.1% or better. In a plot of absorbance as y versus concentration as x, the usual assumption is legitimate. Hence: (a) Always tabulate as y the quantity which has the larger expected random scatter. (b) If both x and y have large scatter, recognize that a more complicated statistical analysis is called for. This type of analysis is rarely required in the Chem 3XX laboratories. ERROR IN THE SLOPE, OR IN x READ OFF FOR A GIVEN y. The Skoog textbook 1 gives formulae (in appendix 1) with which you can calculate not only the slope of a line, but also the standard deviation (i.e. uncertainty) of that calculated slope. Treat this just like the standard deviation of a mean: there is a 68% chance that the slope lies within " one standard deviation of the calculated value. If you wish to use a different confidence level, scale the standard deviation via: ܥܮ ൌ ݔҧ േ ݐݏ √݊ Often, having calculated the line, one wants to take a particular y value (e.g. absorbance of an unknown solution) and read the corresponding x (concentration) from the graph. Your textbook also gives formulae to calculate the uncertainty in this x value. Note that the calculation differs depending if your graph is of a calibration curve or a standard addition line. VI. TESTS OF STATISTICAL SIGNIFICANCE Significance testing is a branch of statistics that applies rigorous statistical procedures based on probability distributions to assess the likelihood of certain “hypotheses”. Each test begins with a null hypothesis and is performed to assess the probability of whether that the null hypothesis is false. For example, if you wanted to test whether two different analytical methods give you statistically different results, the null hypothesis is that there is no difference. You would perform a t-test (below) to see whether there is evidence to reject the null hypothesis. The Grubbs Test The Grubbs test may be used if a series of replicate results has an outlier; that is, one result that is substantially different from the others. This outlier may be due to an error in that single measurement or due to statistical variation. If the latter, the outlier must be retained, if the former, the outlier may be rejected. The Grubbs test determines whether the outlier is statistically different from the others and should be rejected. As a result, this test is applied to the most extreme value in the data set, and only one value may be rejected based on the Grubbs test. The test cannot be used sequentially on a series of outliers. The test is: ܩ ൌ ฬ ݔ ௦௨௦௧ െ ݔҧ ݏ ฬ 36 Where ݔ ௦௨௦௧ is the suspected outlier, ݔҧ is the mean of the data set including the suspected outlier, and s is the standard deviation of the data set. The calculated G value is then compared to the G value from a table at a selected confidence level. If the G calc is smaller than the G table value, the data point is retained; however, if G calc is greater than G table , the data point is considered an outlier and is therefore discarded. The mean and standard deviations are then recalculated, excluding the now rejected outlier. The F-test The F-test is used solely to determine whether two variances (the square of the standard deviations) are significantly different or whether the difference is within normal statistical variation. In the F-test equation, the variance in the numerator is always the greater of the two being tested (s ଶ s ଵ ), so that the F-value is always greater than one. When the F-value is calculated, it is compared to a table value at a selected confidence level. If F calc is greater than the F table , there is reason to believe that the two variances are significantly different. ܨ ൌ ሺݏ ଶ ሻ ଶ ሺݏ ଵ ሻ ଶ Note that result from the F-test only applies to variance of the experimental means; whether two experimentally-derived means are significantly different is determined by the Student’s t test. An application of the F-test would be when a sample is analyzed by two different methods or by two different analysts using the same method (see case 2 below). The variances of the two results should be compared to determine whether they differ significantly. If the two variances are not different, then the two means can then be tested using the student’s t paired test (see below for case 2: Student’s t paired). If the F-test shows that the two variances are different, and the two means need to be tested using a modified Student’s t paired test (see below for case 2: paired test). The Student’s t-test The Student’s t-test is a commonly-used statistical tool that determines whether two results are significantly different at a certain confidence level. The t-test is used to compare results. There are three common cases of the t-test. In all of the t-tests, a t-value is calculated. This t calc is then compared to the t table at a desired confidence level to determine whether the two results are significantly different. Case 1. Comparing a Measured result with a “known” value This case of t-test is utilized if an experimental result is to be tested against a known, accepted or established value. For instance, if a new method (or new instrument or new analyst) is to be tested, it is usually to analyze a sample that contains a known amount of analyte. Samples with certified amount of analyte can be obtained from various sources, notably the (American) 37 National Institute of Standards and Technology, (NIST). The experimental mean (ݔҧ ) then can be compared to the known value (ߤ) to determine whether the new method gave an acceptable answer. In this case, the following equation is used to calculate the experimental t value. ݐ ൌ ሺݔҧ െ ߤሻ√ܰ ݏ In the case of an accepted value, it is assumed that the known value (µ) is exact, so that standard deviation (s) and number of measurements (N) are determined from the experimental data. Note replicate measurements are required or else you won’t have a value for s and N will be zero. The t calc is then compared to t table at a desired confidence level and N-1 degrees of freedom. If the calculated value is less than the table value, the two results are not significantly different (null hypothesis is retained). Case 2. Comparing Replicate Measurements. A slightly more complex case arises if two experimental values are to be compared, for instance if the same sample is analyzed by two different methods (or on two different instruments, etc). The F-test must be carried out before comparing replicate measurements with the t-test because the F-test indicates which t-test to use. a) Case 2: Student’s t-paired test (two ݏ ଶ are similar) In this case, there are two standard deviations and two N values, and weighted averages must be used. As shown in the following equation. The pooled standard deviation (s ୮ ) is given by the second equation, where N is the total number of measurements combined from all methods. Student’s t paired: ݐ ൌ ሺݔ ଵ തതത െ ݔ ଶ തതതሻ ට ಿ భ ಿ మ ಿ భ శಿ మ ௦ ݏ ൌ ඨ ݏ ଵ ଶ ሺܰ ଵ െ 1ሻ ݏ ଶ ଶ ሺܰ ଶ െ 1ሻ. . . ݏ ଶ ሺܰ െ 1ሻ ܰ െ ݇ This kind of paired test, where two analytical results are compared, arises quite often in analytical chemistry. A classic use is when one analyst measures two different samples on one instrument and wishes to compare the samples. 38 b) Case 2: Student’s t-paired test (two ݏ ଶ are not statistically-similar) The following equation is applied when two experimental means are to be compared, but the two have significantly different variances (as determined from F-test). Paired test (two ݏ ଶ are not the same): ݐ ൌ |௫ భ തതതതି௫ మ തതതത| ට௦ భ మ ே భ ⁄ ା௦ మ మ ே మ ⁄ Where degrees of freedom = ൝ ሺ௦ భ మ ே భ ⁄ ା௦ మ మ ே మ ⁄ ሻ మ ሺೞ భ మ ಿ భ ൗ ሻ మ ಿ భ శభ ା ሺೞ మ మ ಿ మ ൗ ሻ మ ಿ మ శభ ൡ െ 2 For Case 2 t-tests, the t-value calculated is then compared with the table t-value at N-k degrees of freedom (where N is the total number of measurements from all methods and k is the number of methods used). This comparison is commonly used to determine whether two instruments or methods or analysts give the same results, and thus determine whether two samples are identical, for example in determining the source of an oil spill by comparing oil from a tanker with oil from a spill. Case 3: Paired t-test for Comparing Individual Differences The paired t test is based on the difference (݀ ) between a pair of results, which are obtained from two different methods. Unlike Case 2 where the same sample is analyzed by two methods, in Case 3, multiple different samples are analyzed by two methods (or instruments or analysts, etc.), so that each sample is associated with two experimental results. Case 3 does not require replicate measurements. The difference (݀ ) between the pair of experimental results from each sample is then used to calculate a mean difference (݀ ҧ ), which is utilized to calculate the t-value. ݏ ௗ ൌ ඨ ∑ሺ݀ െ ݀ ҧ ሻ ଶ ݊ െ 1 ݐ ൌ ห݀ ҧ ห ݏ ௗ √݊ Where n is the number of samples, and ݏ ௗ is the standard deviation based on the differences. 39 The calculated t-value is compared to the table t-value at a selected confidence level and at degree of freedom equal to n-1. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; appendix 1. 2. Harris, D.A. Quantitative Chemical Analysis, 8th ed.; W.H. Freeman: New York, 2010; chapters 3 (experimental error) and 4 (statistics). 40 A-03 Determination of Lead by Anodic Stripping Voltammetry LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the method of standard addition and identify its strengths Describe the purpose of the internal standard Describe the processes happening at the Hg electrode during the deposition and stripping phases of the experiment Describe the differential pulse method and explain how it improves the signal-to- noise ratio of the data Recognize the safety concerns associated with using a potentiostat and in handling nitric acid solutions and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of lead in your unknown making appropriate use of dilution factors, and the standard addition and internal standard approaches Apply fundamental statistics to collected data and calculate the signal detection limit and minimum detectable concentration Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks) through practice BACKGROUND Anodic stripping voltammetry is a two-step electrochemical process in which a negative potential, measured vs. a saturated calomel reference electrode, is first applied to a hanging mercury drop electrode (HMDE). In this step the HMDE acts as a cathode and metal analytes from the solution plate as amalgams onto the surface of the mercury drop. The applied potential is then scanned in a positive direction. Each metal is oxidized back into solution at a characteristic dissolution potential. In this “stripping” process the mercury drop acts as the anode. The current flowing through the HMDE is measured during the stripping process and is the sum of the capacitive current required to charge the ionic layer adjacent to the mercury, and the faradaic current associated with the oxidation of the analyte metal(s). A differential pulse technique is used to subtract the capacitive component of this current. In the differential pulse method, voltage pulses are superimposed onto the voltage ramp that is applied to the mercury drop. The electrode current is sampled just before the pulse and again just before the end of the pulse. The current difference representing primarily the faradaic current is recorded vs. applied potential as a “differential pulse” voltammogram. The current is 41 proportional to the amount of analyte plated onto the mercury drop, and hence to its concentration in the original solution. An aliquot of cadmium is added to the cell at the beginning of the experiment and serves as an internal standard to compensate for variations in plating technique. The height of the peak for lead is compared to that for the cadmium present in constant amount throughout the experiment. A similar comparison can be made with peak area rather than peak height. Anomalies resulting from the emptying and filling of the cell are avoided using the standard addition technique. A voltammogram is run of an aliquot of the unknown lead solution added to a supporting electrolyte of dilute nitric acid. The acid maintains the pH< 2 to avoid the adsorption of lead onto the surface of the glass cell. An aliquot of standard lead solution is then added to the cell and a second voltammogram is run. The procedure is repeated for several further additions of standard lead solution. PROCEDURE Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few millilitres of deionized water and add the washings to the flask. Dilute to the mark with deionized water. Mix well. Empty the cell by aspiration without dismantling. Rinse the cell with deionized water and empty. Rinse with supporting electrolyte and empty. Add 50 mL of supporting electrolyte to the cell. Pipet in 1.00mL of a solution containing 1.00E-04 g/mL of cadmium. Refer to the appropriate instructions following, and use a PAR 174A polarographic analyzer to record three (3) anodic stripping voltammograms of the blank solution containing supporting electrolyte plus internal standard. Pipet in 0.500 mL of diluted unknown solution and run two similar voltammograms. Repeat for the same solution to which has been added 0.500 mL of a standard solution containing 1.00E-04 g/mL of Pb. Add 0.500 mL of standard solution 3 more times to your solution (for a total of 4 additions of standard solution) and run voltammograms between each addition. Take care to repeat each measurement twice. At the end of the experiment you should have recorded at least 13 voltammograms: 3 (blank), 2 (unknown), and 8 (unknown + standard). Each standard addition should be of 0.500 mL for a cumulative volume of 0.5, 1.0, 1.5 and 2.0 mL. WARNING: Potentiostat is capable of producing lethal current and/or voltage. Making of electrical connections must only be done with potentiostat ‘OFF’ or with ‘DUMMY CELL’ selected. Nitric acid solutions are corrosive and act as strong oxidizers. Avoid skin contact. Rinse immediately with cold water if it occurs. 42 OPERATING INSTRUCTIONS FOR THE PAR174 POLAROGRAPHIC ANALYZER Set the following initial parameters: Power On Selector Off Button Initial Initial Potential -0.9 V Potential Scan Rate +5 mV per sec. Range 1.5 V Modulation Amplitude 25 mV Operating Mode Differential Pulse Low Pass Filter Off Output Offset Off Display Direction “-“ Current Range 0.05 mA Data acquisition is through custom-written program using LabVIEW™. Double-click the ‘PAR174_echem’ icon on the desktop. The program has been designed to show a front panel similar to that of the PAR174: set the ‘virtual’ knobs and switches to match the settings above. Pay particular attention to the ‘Initial Potential’ and ‘Potential Scan Rate’ settings as these are used to calculate the electrode potential during the experiment. Turn on the N 2 supply to the cell (note regulator has two knobs: one opens the cylinder, the second allows the gas to flow into the lines). Adjust the regulator pressure to ~15 psi. Set the electrode system to operate in the HMDE (hanging mercury drop electrode) mode and set the drop size to "3". Leave the deaeration lever on the electrode system at "O". Dispense two mercury drops with the "MAN" switch on the electrode system (the first drop serves to flush the capillary). Set the stirrer speed to "2", start the timer, and allow the solution to mix for 1 minute. Start deposition by setting the SELECTOR switch to EXTERNAL CELL thus applying a -0.90 V potential to the mercury drop. After 100 sec. plating, turn off the stirrer then wait 20 sec. to allow the diffusion layer near the drop to stabilize (i.e. for mixing to cease). To record the voltammogram, simultaneously press the SCAN button on the potentiostat and start the LabVIEW software (arrow button in top left). The scan is complete once you have scanned to -0.30V. In LabVIEW, depress the rectangular ‘STOP’ button to end data acquisition and save your datafile. If you instead click the stopsign button, your data will not be saved. Set the SELECTOR switch on the potentiostat to OFF and depress the INITIAL button. Change the ‘filename’ or ‘filename number’ in LabVIEW in readiness for the next run. To repeat the run it is not necessary to remove the solution from the cell. Dispense a fresh mercury drop, make any necessary changes to the instrument or software settings, and repeat the plating and the scan as before. 43 After the last run empty the cell by aspiration. Rinse with deionized water and refill with supporting electrolyte. Leave the cell in this condition with a mercury drop attached to the capillary. Determine the concentration in g/mL of lead in your original unknown solution as received in the vial. IN YOUR REPORT Describe the principles and application of both the standard addition and the internal standard methods. Consider how the use of the internal standard corrects for plating variations – how should you process your data? Describe how the differential pulse technique isolates only the portion of the signal due to the analyte. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 25D-E. 2. Harris, D.A. Quantitative Chemical Analysis, 8th ed.; W.H. Freeman: New York, 2010; chapters 17-5 and 5-2. 3. Bard, A.J.; Faulkner, L.R. Electrochemical Methods: fundamentals and applications; Wiley: New York, 2001; chapters 6 and 7. (QD553 .B37 2001 in IKBLC) 44 A-04 Determination of Iron and Chromium by Spectrophotometry LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the multiwavelength linear regression analysis method Describe Beer’s law and its limitations Describe the advantages and disadvantages of single-beam and double-beam spectrophotometers Describe the advantages and disadvantages of using photodiode array and photomultiplier tube detectors Recognize the safety concerns associated with the use of UV light sources and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of iron and chromium in your unknown Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks) through practice BACKGROUND Solutions containing mixtures of two non-interacting species can be determined spectrophotometrically by multiwavelength linear regression analysis (MLRA) provided the analytes have highly-overlapped UV-visible spectra. Absorbances are measured at several wavelengths in the overlapping spectral region for the unknown mixture and the standard solutions containing the individual species. By Beer's law the absorbances of the standards, A s1 and A s2 , are: A s1 = ε 1 bc s1 (1) and A s2 = ε 2 bc s2 (2) Where b is the cell path length, and ε 1 and ε 2 , and c s1 and c s2 are the molar extinction coefficients and molar concentrations, respectively, of each species. The absorbance A m of the mixture is: A m = ε 1 bc 1 + ε 2 bc 2 (3) Where c 1 and c 2 are the molar concentrations of the components. Substitution for ε 1 and ε 2 from eq'ns (1) and (2) into eq'n (3) gives: 45 A m /A s1 = c 1 /c s1 + (c 2 /c s2 )(A s2 /A s1 ) (4) Thus a plot of A m /A s1 vs. A s2 /A s1 gives a straight line. The concentrations of species 1 and 2 are calculated from the intercept and slope respectively. The method is used for the simultaneous determination of iron and chromium. Acid solutions of the unknown mixture and the individual standards have been prepared for you. Absorbances are measured in the spectrally overlapped 240 - 370 nm region. You will make duplicate analyses are made with a PE124 double beam spectrophotometer and a Hewlett-Packard 8452A diode array spectrophotometer. The PE124 measures the ratio of the intensity of light transmitted by the sample solution to that transmitted by the reference solution. In this way it compensates for the spectral characteristics of the lamp and the detector. In this experiment absorbances will be recorded at 10 nm (minimum) intervals. The HP8452A spectrophotometer uses an array of 328 light detecting diodes to simultaneously monitor light intensities at 2 nm intervals over the complete spectral range. As this is a single beam instrument, a lamp spectrum is first measured with a blank solution in the cell compartment. The blank spectrum is stored by computer, then the spectrum of the sample solution is recorded and the absorbances are calculated as a function of wavelength. A complete spectrum is recorded and plotted in less than a second. Note: your accuracy in this experiment depends strongly on the quality of your solution preparation. PROCEDURE Transfer all of your unknown solution to a 100mL volumetric flask. Note that the unknown provided is made up in 0.1M H 2 SO 4 . Rinse the vial with a few mL of deionized water and add the washings to the flask. Dilute to the mark with water. Mix well. Using the iron stock solution (2.00E-04 g/mL iron in 0.1M H 2 SO 4 ), prepare 100mL of 2.00E-05 g/mL Fe solution. Using the chromium stock solution (1.93E-04 g/mL in 0.1M H 2 SO 4 ), similarly prepare 100mL of 1.93E-05 g/mL Cr solution. Prepare a blank solution of 0.01M H 2 SO 4 solution using the 0.2M H 2 SO 4 stock. Use the Perkin Elmer 124 spectrophotometer to measure the absorbances of the standard solutions and of the mixture. Take readings at least every 10 nm from 240 - 370 nm. Recording more measurements should result in increased accuracy. Turn on POWER SUPPLY. Wait 15 seconds. Turn on the D 2 lamp. Set the VISIBLE-ULTRAVIOLET lever (left side of instrument) to ULTRAVIOLET. Also power on the instrument itself (‘Operation’ knob on front panel). Set wavelength anywhere between 240 nm and 370 nm. WARNING: UV light can be harmful because it is energetic enough to break bonds and thus harm tissue (skin, eyes, etc). The best protection is avoidance. Polycarbonate safety glasses block most UV. 46 Switch ABS/%T to %T. Insert a 0%T shutter into the SAMP. cell holder and use ZERO CHECK (right side of instrument) to zero the meter. NOTE: HANDLE THE CELLS CAREFULLY – they cost $500(!) per pair because they are spectrally-matched. The solutions contain sulfuric acid. Take care to avoid spills when filling the cells and inserting them into the cell compartment of the spectrophotometer. Two-thirds fill two cells with blank solution and insert into the SAMP. and REF. cell holders. When inserting the cell, always keep the same orientation and try to position the cell reproducibly. Orient the clear windows towards the right and left. Set ABS/%T to ABS. Set absorbance SCALE to 0-1. Zero the meter with the front panel ZERO knob. Do not change this setting for the rest of the experiment. Measure the absorbances of the blank solution at 10 nm intervals between 240 and 370 nm. Replace the blank solution in the sample cell with iron standard, chromium standard, or unknown mixture, and measure the absorbance every 10 nm between 240 and 370 nm. Calculate the concentrations of iron and chromium in your original unknown solution as contained in the vial. Repeat the experiment using a Hewlett Packard 8452A diode array spectrophotometer to measure the absorbances of the standard solutions and of the unknown mixture. HP 8452A Instrument control and acquisition of data: Make sure there are no cuvettes in the cell holders, then turn on the spectrophotometer power (switch at rear of instrument). The spectrophotometer must be powered on before initializing the computer software. Wait until the LAMP indicator on the instrument confirms the lamp has been ignited, do not turn the deuterium lamp on and off unnecessarily! This shortens the life of the lamp (~$1000)! Allow 15 min. for the lamp to warm-up and its intensity to stabilize before making measurements. Double click the Olis GlobalWorks icon to load the software, then select HP Diode Array under the Data collection tab, click open. Check that these parameters are set: Under Live Display tab, set wavelengths to Scan from 240 nm to 410 nm Under Repeated Scans tab, set number of scans to 1 Rinse and fill the cuvette (2/3 full) with blank solution. When inserting the cell, always keep the same orientation and try to position the cell reproducibly. Click 47 Collect Ref. The background will be subtracted automatically from all subsequent spectra. Rinse and fill the cell (2/3 full) with sample solution. Insert the cuvette into the cell compartment using the same orientation as for the blank. Click Collect Data. Click yes to transfer data into GlobalWorks. To change the file names, double click the Name option in the lower right window as shown here at right: If graph displays a 3D view of the data: from the tool-bar menu, select View and then 2D view (Double click graphs to expand). Run similar scans for the remaining standard solution and the unknown. To collect data for the next sample, click the HP Diode Array on the right hand side of the screen and then collect data. The graphs for subsequent runs will be displayed under the Experiment window as shown below: To overlay sample spectra, open one graph from under the experiment window, click the graph area so that the graph turns yellow. Then, select Edit from the menu bar and then Copy slice. Open the second graph, click graph area, select Edit and Paste slice. Select View and then 2D View. The overlay graph will be created as a new file. Double click on overlay graph area to expand graph, select File, Print to print overlay spectra. To save overlay graph and data in GlobalWorks, click the overlay graph, File and Save dataset as into the folder CHEM 3XX A4 DATA located on the desktop. Save the file in the format: StudentNames_Sample name. NOTE: only the overlay data and graph will be saved! Right click the overlay graph, select Export data to Excel. The first column displays the wavelengths and the column to the right display absorbance values (all overlay data are exported). Name the Excel file with student name and sample identification. Processing data with Microsoft Excel: Note: You are assumed to be familiar with the use of Microsoft Excel for data analysis and plotting graphs. Helpful tutorials can be found on the internet if you need a refresher. 48 The spreadsheet will display columns of data labelled ‘0’, ‘1’, ‘2’, and ‘3’. The leftmost should be readily identifiable as your wavelength in nm. The other three represent absorbance of your samples. The identity of each is determined by the order in which you copied/pasted the data before exporting it to Excel. If you have ANY doubt about which is which, you should export the spectra to Excel individually. In this case, you should first label the absorbance columns in the three files, and then combine the three spectra into one spreadsheet. Choose which absorbances will be A s1 and which will be A s2 and set up a column in which A s2 /A s1 is calculated for each wavelength. Set up another column in which A m /A s1 is calculated. Use the Excel plotting functions to plot graphs of the spectral overlay and A m /A s1 vs. A s2 /A s1 in the 240 nm to 370 nm spectral region. Use the linear regression function to calculate the slope, intercept, and the associated 95% C.L. values. Calculate the concentrations of iron and chromium in your original unknown solution as contained in the vial. Note a common mistake is to mix up which species you gave the designation ‘1’ and which ‘2’; this reverses the results for Fe and Cr. IN YOUR REPORT Compare the results obtained with the two instruments. In comparing your results, make sure to use the appropriate statistical tests to determine whether the results agree. Discuss the advantages and disadvantages of each instrument. Identify one result for the iron analysis and one for the chromium analysis to be marked. This could be the result from the HP instrument, or from the PE instrument, or an average of the two. Rationalize your choice. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapters 13 and 14. 2. Harris, D.A. Quantitative Chemical Analysis, 8th ed.; W.H. Freeman: New York, 2010; chapters 17, 18 and 19. 3. Blanco, M.; Iturriaga, H.; Maspoch, S.; Tarin, P. J. Chem. Ed. 1989, 66(2), 178. 4. Perkampus, H.-H. UV-Vis spectroscopy and its applications; Springer-Verlag: New York, 1992. (QD96.U4P4713 1992 in IKBLC) 49 A-06 Determination of Quinine by Fluorometry and Absorbance Spectrometry LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the processes of absorbance and fluorescence Describe how absorbance and fluorescence intensity vary as a function of analyte concentration Describe the advantages and disadvantages of absorbance and fluorescence measurements Describe the differences and similarities between an absorbance spectrum, excitation spectrum and emission spectrum Recognize the safety concerns associated with using UV light sources and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of quinine in your unknown using the calibration curve method and applying appropriate dilution factors Describe the purpose of the various pieces of the modular fluorimeter and explain their particular placement Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks) through practice BACKGROUND Quinine is an alkaloid that occurs naturally in the bark of the chinchona tree. It was widely-used medicinally as an antimalarial agent from about 1800 to around 1940 when more effective treatments were discovered. It was commonly administered as a solution in water, with sugar added to mask the bitterness of the quinine. This solution became known as tonic water (a ‘tonic’ is a liquid medicine). In Canada, tonic water contains quinine as a flavouring agent with a maximum permissible concentration of 83 ppm quinine. Owing to its cyclic aromatic moiety, quinine is strongly fluorescent in acid solution. In this experiment quinine is analyzed both in an unknown solution and in a sample of tonic water by absorbance spectrometry and fluorometry. Absorbance spectrometry: Transmittance of light through a solution is defined as I/I 0 , where I 0 is the initial intensity of the light, and I is the intensity after it has passed through the solution. The absorbance A of the solution is given by: | | ¹ | \ | − = − = 0 I I log T log A (1) 50 In practice, I 0 is usually redefined as the intensity of light passed through an optical cell containing blank solution. Eq’n (1) then gives the absorbance of an analyte without further correction for the absorbances of the solvent or the cell. The Beer-Lambert law, bc A ε = (2) where ε is the molar extinction coefficient, b is the cell path length, and c is the molar concentration of the analyte may then be applied. An unknown concentration can therefore be interpolated from a calibration plot of absorbance vs. concentration for a series of standard solutions. Fluorometry: Molecular fluorescence is a process by which molecules first absorb electromagnetic radiation, then re-emit the absorbed energy as light. The emitted light is of longer wavelength than the incident light, and each photon emitted goes in a random direction. The intensity I em of the emitted light is given by: ( ) bc 0 em 10 1 P K I ε − − ′ = (3) where K′ is a function of the quantum efficiency of the emission process. Equation (3) can be expanded as the series: ( ) ( ) ( ¸ ( ¸ ⋅ ⋅ ⋅ ε + ε − ε ′ = ! 3 bc 303 . 2 ! 2 bc 303 . 2 bc 303 . 2 P K I 3 2 0 em (4) At low concentrations eq’n (4) is linearly-approximated as: Kc bc 303 . 2 P K I 0 em = ε ′ = (5) Therefore the concentration of an analyte can be interpolated from a calibration plot of I em vs. c for a series of standard solutions. I em is usually measured at right angles to the direction of the excitation beam to minimize detection of any scattered light from the source. Also, the light from the source is passed through a bandpass filter or monochromator that transmits shorter wavelengths suitable for excitation, while eliminating longer wavelengths that may be scattered and detected with the emitted light. Fluorometry is in principle more sensitive than absorptiometry, since the measured signal approaches zero as the concentration of the emitting species approaches zero. At low concentrations the emission signal can be increased by increasing the intensity of the source or by using a more sensitive detector; for instance, a photomultiplier tube instead of a CCD array. 51 On the other hand, when measuring low concentrations by transmittance or absorbance, the measured signal approaches a maximum as I approaches I 0 (eq’n 1) and T approaches 1. It can be shown 1 that the relative standard deviation in concentration, s c /c, is related to the absolute standard deviation of transmittance, s T , by: T log T s 434 . 0 c s T c = (6) Therefore, as T Y1, s c /c Y 4 . To improve the sensitivity of analysis by absorbance it is necessary to reduce s T , which is a function of instrument noise, signal drift, etc. Absorbance and fluorescence spectra: A plot of absorbance vs. wavelength is a function of the ratio of I to I 0 . Therefore, although I and I 0 are functions of instrument parameters such as lamp intensity and detector response, both of which vary with wavelength, the effects of the instrument parameters cancel in the process of calculating the ratio. The resulting absorbance spectrum is characteristic only of the absorbing species and is independent of instrument parameters. A fluorescence excitation spectrum is a plot of the intensity of emitted light I em vs. the wavelength of the excitation light, 8 ex . This spectrum is similar to an absorbance spectrum, and in fact an absorbance spectrum can be used instead of an excitation spectrum to determine wavelengths suitable for excitation. A fluorescence emission spectrum is a plot of the intensity of emitted light I em vs. emission wavelength 8 em . Unlike plots of absorbance vs. wavelength, measurements of emission intensity as a function of 8 em give spectra which are characteristic not only of the analyte but also of wavelength-dependent instrument parameters. If the detector gain is higher at a given wavelength, the measured signal will be higher. Corrected spectra can only be constructed by referencing the measured plots against a known emission spectrum such as the light emitted from a black body source. Of the two fluorimeters you will use in this experiment, one can automatically correct the spectra using parameters determined at the factory, while the other requires manual calibration. This manual calibration is against light from a reference source: a halogen lamp with a colour temperature of 3500K. The light from the lamp is measured and the intensity I W,8,meas is stored as a function of λ. The stored data is then compared with a data file of the actual spectrum of the lamp, λ , W I vs. λ, and at each wavelength a correction factor B 8 is calculated, where: λ λ λ = , W meas , , W I I B (7) A corrected emission spectrum of the analyte, (I em /B 8 ) vs. 8 em is then plotted. This plot is characteristic of the analyte, and is independent of instrument parameters. Instrumentation: This experiment compares both absorbance spectrophotometry with fluorescence spectrophotometry and inexpensive instrumentation with top-of-the-line instrumentation. You will use an Ocean Optics spectrometer capable of both absorbance and fluorescence measurements as well as the Agilent Cary Ecilpse Fluorimeter and the Agilent Cary 52 5000 UV-Vis spectrophotometer. These last two instruments are located in the departmental shared instrument facility (SIF), room B353. The Ocean Optics spectrometer is a good example of the current trend towards miniaturization. Although the instrument is tiny – about the size of a pack of cards, it incorporates a grating monochromator and a linear CCD array detector. The working wavelength range of the instrument is roughly from 250 to 910 nm and gives resolution of about 1.3 nm. The modular design of the instrument allows you to insert different gratings that can improve the resolution but with a loss of wavelength range. This modular design also means it can be configured to measure absorbance or fluorescence. Light from either a deuterium/tungsten or pulsed xenon source is monitored. An optical fiber transmits light from the source to the cell and on to the spectrometer. For absorbance measurements the monitored light is that transmitted through the cell, while for fluorescence measurements the light detected is that emitted at right angle to the direction of the incident beam. By contrast, the Cary 5000 is a double-beam spectrophotometer with two monochromators and two detectors: a photomultiplier tube for UV-Vis detection and a lead sulfide photocell for NIR detection. It offers vastly higher resolution (0.05 nm) and a much larger working range: absorbance values up to 8 are measurable. The instrument covers the 190 – 3300 nm wavelength range; from deep UV to near infrared. The Cary 5000 also costs about 30x more than the Ocean Optics instrument! The Cary Eclipse Fluorimeter is similarly top-of-the-line. It offers a broader working range, higher sensitivity, better signal-to-noise ratio, and more user customizable parameters than the Ocean Optics instrument. The Eclipse costs roughly 10x more. PROCEDURE (Before preparing your solutions, proceed to the SIF in room B354 and power on the Ocean Optics UV source (keep shutter closed) and power on the Cary 5000 UV-Vis and Cary Eclipse instruments. Return to C224 to make your solutions while the instruments warm-up. Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few millilitres of water and add the washings to the flask. Dilute to the mark with water. Mix well. Suspend a flask containing 20 - 30 mL tonic water in an ultrasonic bath for about two to three minutes to remove the carbonation. Dilute your degassed tonic water 25-fold using volumetric glassware. Using 1.00E-05 g/mL quinine stock solution prepare at least five (preferably six) calibration standard solutions. Start with zero concentration (blank) and do not exceed 1.25E-06 g/mL quinine for the most concentrated solution. Prepare further five-fold dilutions of your tonic water solution and your unknown. All your final solutions (calibration, tonic water, unknown) should be in 0.04M sulfuric acid. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 50mL volumetric flasks are provided. Ensure each solution is well mixed. You should arrive in the laboratory with a plan of how to prepare all of these solutions! 53 Bring your samples to the SIF and record the UV-Vis absorbance spectrum of your most concentrated stock solution and of your diluted tonic water sample. Print these two spectra. Determine the wavelength of maximum absorption for your stock solution, and then record the absorbance at this wavelength for each of your solutions. APPENDIX I gives operating instructions for the Cary 5000 instrument. Return to the analytical lab to proceed with fluorescence measurements on the Ocean Optics instrument. With the instrument configured as in Fig. 1 the optical fiber monitors light transmitted through the sample so that transmittance and absorbance spectra can be recorded. Refer to APPENDIX II sections B and C and measure the transmittance of the OF2U330 bandpass filter that will be used in making fluorescence measurements. Print the spectrum – you will need to comment on it in your report. Turn off the deuterium lamp and reconfigure the spectrometer as in Fig. 2a. Refer to APPENDIX II section D(i) to measure a reference emission spectrum of the light from a black body source (halogen lamp). The spectrometer monitors light from the source reflected at 90˚ by a glass plate. Reconfigure the spectrometer as in Fig. 2b, then refer to APPENDIX II section D(ii) to record corrected emission spectra of the standard solutions, the diluted unknown, and the diluted tonic water. Print uncorrected and corrected emission spectra of the most concentrated stock solution, and the corrected emission spectrum of the tonic water. Do not print spectra for the other solutions. Use the cursor function to identify the wavelength of the emission maximum for quinine and record the emission intensities of the standards and unknown solutions at this wavelength. Return to the SIF to make similar fluorescence measurements on the Cary Eclipse instrument. Use the wavelength of maximum absorption that you determined previously as your excitation wavelength. Record and print corrected fluorescence emission spectra for the most concentrated stock solution and for the diluted tonic water sample. Record the intensity of fluorescence emission for all samples. APPENDIX III gives operating instructions for the Cary Eclipse. Calculate the concentrations of quinine in your original unknown solution by absorbance and fluorescence. Decide which of your two fluorescence determinations is likely more accurate. Comment on the reasons for your decision in your report. Comment on the meaning of your measured concentration of quinine in tonic water. On the coverpage of your report, include the analytical result determined by absorbance and by fluorescence. Report only one of your two fluorescence results. WARNING: UV light can be harmful because it is energetic enough to break bonds and thus harm tissue (skin, eyes, etc.). The best protection is avoidance. Special UV-blocking goggles must be worn when connecting or disconnecting fiber optic cables if the lamp is ‘ON’. 54 IN YOUR REPORT Explain the phenomena of absorbance and fluorescence. Discuss the transmission spectrum of the U-330 filter and its importance in your experiment. Discuss the difference between the corrected and uncorrected emission spectra of quinine. Compare the absorbance and emission spectra of the tonic water with those of the quinine standards. Comment on the effect, if any, that the solution matrix may have on the analysis of quinine in tonic water by absorbance and fluorescence. Compare the analysis of quinine by these two methods. In comparing your results, make sure to use the appropriate statistical tests to determine whether the results agree. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6 th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 15. 2. Harris, D.A. Quantitative Chemical Analysis, 8 th ed.; W.H. Freeman: New York, 2010; chapters 18-5 and 18-6. 3. Lakowicz, J.R. Principles of Fluorescence Spectroscopy, 3 rd ed.; Springer: New York, 2006. (Available online through UBC library ebrary service) 55 APPENDIX I: Operating Instructions for the Agilent Cary 5000 UV-Vis Spectrometer Ensure the instrument is powered on (switch on front). Open the instrument software from the computer desktop: Open the ‘Cary WinUV’ folder, then double-click the ‘Scan’ icon. Choose ‘Setup’. In the dialog box, set the following parameters: Under the ‘Options’ tab, enter: Under the ‘Baseline’ tab, select: SBW (spectral bandwidth): 5 nm Baseline Correction Beam: Single front Energy: 1 Slit height: full UV/Vis source changeover: 350 nm Then click ‘OK’ Fill a quartz cuvette two-thirds full with your blank solution and insert the cuvette into the front position of the spectrometer, ensuring the clear windows are oriented left-right. Zero the instrument (button on left of screen), then click ‘Baseline’ to acquire a baseline. The system will 56 prompt you to insert a blank sample. Click ‘OK’ to measure the baseline. Once complete, remove the cuvette and two-thirds fill it with your most concentrated stock solution. Click ‘Start’ to begin your measurements. Note that the graph may show this absorbance as being higher or lower than that of the baseline; this is fine as the instrument has set the reference point to zero before measuring your sample. Data is saved automatically; use the standard Windows print functions to print your spectrum. Repeat with your diluted tonic water solution. Close the software after recording your spectra. Again in the ‘Cary WinUV’ folder, double-click the ‘Concentration’ icon. This will launch a program that will record absorbance at a specified wavelength, build a calibration curve and provide (partial) results. Similar to before, click ‘Setup’ and enter the parameters for your measurements. Under the ‘Cary’ tab, enter the wavelength of maximum absorption that you determined from the scan of your standard solution. The number of replicates should be one, the bandwidth (SBW) 5 nm, and the averaging time 1s. Other parameters should be as you set previously. In the ‘Standards’ tab, make settings similar to the ones below but provide your own concentration values for each standard solution and adjust the units to suit. Don’t forget to include your blank solution as a standard! In the ‘Samples’ tab, enter two as the number of samples and fill in the table with names identifying your two samples (diluted unknown and diluted tonic water solutions). Click ‘OK’. Under ‘Options’, set the beam mode to single beam (front). 57 Click the ‘Start’ button to begin the measurements. The software will prompt you to insert each sample sequentially, will build your calibration curve and will directly output the concentration in your diluted unknown and your diluted tonic water sample. If any of the datapoints on your calibration curve are problematic, you can remeasure the datapoint (button at bottom-left of screen). If it is still problematic, manual treatment of your data will be required. Print the report the software provides you. 58 PULSED XENON SOURCE DH2000 D2 SOURCE USB2000 SPECTROMETER TO USB PORT OF COMPUTER 0.25m 450 MICRON FIBER 0.25m 450 MICRON FIBER 1 meter 450 MICRON ATTENUATOR LENS LENS CELL APPENDIX II: Operating Instructions for the Ocean Optics USB2000 Spectrometer A. Configuring the USB2000 (i) Configuring the spectrometer to measure transmittance and absorbance: The spectrometer monitors light transmitted through the source fiber and the cell to the detector fiber. The DH2000 deuterium source is used for measurements in the UV spectral region. The attenuator reduces the intensity of the transmitted light to within the dynamic range of the instrument. The halogen lamp of the DH2000, and the pulsed xenon source are turned off while measuring transmittance or absorbance. Fig. 1: Configuration for measuring transmittance and absorbance (ii) Configuring the spectrometer to measure a reference spectrum of the emission from a black body source (tungsten lamp): Light from the DH2000 halogen source is transmitted via 450 micron fibers (red colour-code) and is reflected at right angles by a 45˚ glass plate inserted into the cell compartment. (The glass plate reflects about 4% of the incident beam towards the spectrometer.) The spectrometer monitors the reference light via a 600 micron fiber (brown colour-code). The deuterium lamp of the DH2000, and the pulsed xenon source, are turned off while the reference spectrum is recorded. Fig. 2a: Configuration for measuring an emission reference spectrum ATTENUATOR USB2000 SPECTROMETER TO USB PORT 600 MICRON 0.25m 450 MICRON FIBER DH2000 HALOGEN SOURCE LENS PULSED XENON SOURCE LENS 45˚ GLASS PLATE MIRROR PLUG 1 meter 450 MICRON 59 (iii) Configuring the spectrometer to measure a fluorescence emission spectrum: The spectrometer controls the triggering of the high intensity xenon source to produce excitation pulses coincident in time with the detection of fluorescence. The light emitted from the sample is measured at right angles to the direction of propagation of the incident beam. A relatively large 600 micron fiber (brown colour-code) increases the sensitivity of detection of the low-intensity emitted light. The mirror plugs further increase sensitivity by reflecting light back through the cell. The bandpass filter minimizes detection of scattered light from the source. Fig. 2b: Configuration for measuring fluorescence emission B. Instrument control: “OOIBase32” program Click the “OOIBase32” program. The following window will be displayed. USB2000 SPECTROMETER TO USB PORT OF COMPUTER 600 MICRON FIBER DT1000 UV/VIS SOURCE MIRROR PLUGS PULSED XENON SOURCE LENS CELL BANDPASS FILTER TRIGGER PULSE 60 You will use the following buttons shown in the “Spectrum 1” window: Scope mode – displays intensity (I 8 ) vs. wavelength (λ) without processing. Transmittance mode – calculates and displays T (eq’n 8) vs. λ. Absorbance mode – calculates and displays A (eq’n 9) vs. λ. Irradiance mode – calculates and displays I em,corr (eq’n 11) vs. λ. Store Reference – used in the Scope mode to store I 0 for the Absorbance and Transmittance modes, and I em,ref for the Irradiance (fluorescence) mode. Store Dark Spectrum – used in the Scope mode to store D λ for the Absorbance, Transmittance, and Irradiance (fluorescence) modes. Autoscale the graph. Manually scale the graph. Unscale the graph. C. Measuring Transmittance of the U330 Bandpass Filter Wear the special UV-blocking safety goggles (in locker drawer) at all times when working with the UV light source. Never look into the exit port of a lamp source or into the end of an optical fiber while the source is turned on! 61 • Configure the spectrometer as in Fig. 1 (APPENDIX section A). Set the “Open-Close- TTL” switch at “Close”. Turn on the deuterium source of the DH2000 lamp supply. • Set “Integ. Time” to 30 msec, “Average” to 10 (averages 10 sets of readings), and “Boxcar” to 5 (averages 125 pixels on either side of the selected wavelength). • Click (Scope mode). Set the “Open-Close-TTL” switch on the lamp to “Open”. Adjust the attenuator disk carefully to set a maximum signal of ~3500 counts in the 300- 400nm region. Allow the lamp to warm up for at least 10 minutes. • With an empty cell compartment (no cell), click to measure and store a reference spectrum of I 0 vs. λ. • Insert a black or gray block into the cell compartment. Click to measure/store a dark spectrum (D λ vs. λ). Remove the block. • Click to switch to the transmittance mode. • Insert the U330 bandpass filter into the cell compartment. Click and rescale the x-axis to 200-800nm. Print the spectrum. D. Measuring Fluorescence (i) calibrating the instrument against a black body source: • Turn off the deuterium source. Configure the spectrometer as in APPENDIX section A Fig. 2(a). Insert the 45˚ glass reflector into the cell compartment. • Click . Set “Integ. Time” to 500 msec., “Average” to 1, and “Boxcar” to 4. • Turn on the “Halogen” source of the DH2000 lamp supply. The beam from the source is reflected 90˚ by the glass plate and the attenuated light is monitored by the spectrometer via the 600 micron fiber. Install the cell compartment cover to prevent detection of ambient light. Carefully adjust the attenuator to reset the maximum intensity to ~3500 counts. • Switch “Average” to 4. • Click to measure and store a reference spectrum of meas , , W I λ vs. λ. • Insert a black or gray block into the cell compartment. Wait until the baseline is flat, then click to store a dark spectrum. 62 • Turn off the DH2000 halogen source. (ii) Measuring corrected fluorescence emission: • Configure the spectrometer with two mirror plugs installed as in Fig. 2(b). Insert the U330 bandpass filter into the filter slot. Click “Shutter Open” (√) and “Strobe/Lamp Enable” (√) to turn on the PX-2 pulsed xenon source. (If you don’t hear the pulsed source when it is turned on, check the on/off switch on the back of the PX-2). • Half-fill the cell with your most concentrated standard solution of quinine and insert it into the cell compartment. The fluorescence emission is visible from above the cell. Click . An uncorrected emission spectrum will be displayed. Install the cell compartment cover. Click to re-scale the y-axis. Print the uncorrected spectrum. • Select “Spectrum”, “Ref. Color Temp.”, and set the color temperature of the reference spectrum to 3500K. Click OK. Then click to display the corrected emission spectrum. Click to re-scale the y-axis. Print the corrected spectrum. Use the cursor function to select the peak wavelength and to read the intensity of the emitted light. Similarly measure the corrected fluorescence emissions of your remaining standard solutions, your diluted unknown solution, and the tonic water. You do not need to print copies of the spectra; just note the emission intensity for each solution. The spectrometer outputs spectral data to a computer for processing. The computer processes the data in one of four ways, depending upon the experiment: 1) Scope mode – Intensity (I 8 ) vs. wavelength (λ) is displayed without any processing. The displayed plot is characteristic of both the sample and the spectral response of the instrument. This raw data is processed by the computer in the Transmittance, Absorbance, and Irradiance (fluorescence) modes. 2) Transmittance mode – The computer calculates and plots vs. λ: % 100 D I D I T % , 0 × − − = λ λ λ λ λ (from eq’n 1) (8) where I 0,8 is the intensity of light transmitted through the blank, I 8 is the intensity of light transmitted through the sample, and D 8 is the dark current measured with a black block inserted in the cell holder. 63 3) Absorbance mode – The computer calculates and plots vs. λ: | | ¹ | \ | − − − = λ λ λ λ λ D I D I log A , 0 10 (from eq’n 1) (9) 4) Irradiance (fluorescence) mode – The emission from a tungsten lamp is first measured and the software calculates and stores as a function of λ: λ λ λ λ − = , W meas , , W I D I B (from eq’n 7) (10) The sample emission is then measured, and the computer calculates and plots vs. λ: λ λ − = B D I I meas , em corr , em (11) APPENDIX III: Operating Instructions for the Agilent Cary Eclipse Fluorimeter Ensure the instrument is powered on (switch on front). Open the instrument software from the computer desktop: Open the ‘Cary Eclipse’ folder, then double-click the ‘Scan’ icon. Choose ‘Setup’ and enter the following parameters: Data mode: Fluorescence Scan setup: Emission X mode: Wavelength, nm Excitation: enter your wavelength of maximum absorption in nm Start wavelength: 300 nm Slit width: 5 nm Stop wavelength: 750 nm Slit width: 5 nm Scan control: Medium. Under the ‘Options’ tab, ensure the PMT detector voltage is ‘Low’ Two-thirds fill your cuvette with diluted unknown solution and insert it in the instrument. Click the ‘Start’ button to begin the measurement. Data is saved automatically; use the standard Windows print functions to print your spectrum. Close the software after recording your spectra. Again in the ‘Cary Eclipse’ folder, double-click the ‘Concentration’ icon. Like on the Cary 5000 UV-Vis instrument, this will launch a program that will record absorbance at a specified wavelength, build a calibration curve and provide (partial) results. Under the ‘Cary’ tab, enter your excitation wavelength as before and enter the wavelength of maximum emission from your fluorescence scan of the diluted unknown solution. Ensure the PMT detector voltage is set to low in the ‘Options’ tab. 64 Use the ‘Standards’ and Samples’tabs to provide the instrument information about your solutions as before. Click the ‘Start’ button to begin the measurements. The software will prompt you to insert each sample sequentially, will build your calibration curve and will directly output the concentration in your diluted unknown and your diluted tonic water sample. If any of the datapoints on your calibration curve are problematic, you can remeasure the datapoint. If it is still problematic, manual treatment of your data will be required. Print the report the software provides you. A-0007 FOR BLMSC STUDENTS ONLY A-0007 Determination of Glutamine by Capillary Electrophoresis Learning Objectives 1. Explain the basis of analyte separation in capillary zone electrophoresis 2. Be able to predict the relative migration times of different analytes; similarly, predict how the degree of ionization of a weak acid will affect its migration velocity. 3. Qualitatively use the Van Deemter Equation to compare the theoretical resolution of CE to other chromatographic methods. 4. Describe the principal components of the CE instrument and outline potential instrumental sources of error. 5. Explain the reasons for using an internal standard; describe the ideal attributes of an appropriate internal standard. 6. Determine the mass of glutamine in your unknown using the calibration curve and internal standard methods. 7. Apply fundamental statistics to collected data and calculate the confidence interval of your result. BACKGROUND Capillary electrophoresis (CE) provides excellent resolution in the separation of biological molecules. This experiment utilizes a basic mode of CE – capillary zone electrophoresis – in the separation of a mixture of amino acids. In the presence of an electric field, separation is achieved based on the different electrophoretic mobilities of the analytes. The electrophoretic mobility is proportional to the charge-to-size ratio of the analyte. The product of the applied electric field and the analyte’s electrophoretic mobility equals the analyte’s electrophoretic velocity : cp . Positively-charged analytes have : cp in the direction of the electric field towards the cathode while negatively-charged analytes have : cp against the direction of the electric field towards the anode; a higher charge-to-size ratio increases the magnitude of : cp . The applied electric field is also responsible for a common electroosmotic flow (EOF) towards the cathode. This is caused by the movement of buffer cations – which are attracted to the negative cathode – and their associated solvation shells that drag along solvent molecules. The flat flow profile of EOF contrasts with the laminar profile of pressure-driven flow as shown in Figure 1. The flat profile results from the buffer cations moving near the negatively-charged capillary surface. The negative charge is due to the ionization of surface silanol groups on the inside wall of the silica capillary; this ionization is forced by use of basic pH buffers. Figure 1: Profiles of flat flow (a) and laminar flow (b) The overall velocity of an analyte is the sum of its unique electrophoretic velocity and the common EOF velocity. Because the EOF velocity is much larger than the electrophoretic velocity, all compounds – even anions – migrate towards the cathode. : o¡c¡uII = : L0P + : cp (1) Both the EOF velocity and the electrophoretic velocity are dependent on the electric field. A large positive electric field will increase EOF velocity while increasing the separation between positively and negatively charged analytes – as the former will acquire a positive electrophoretic velocity on top of the EOF velocity and the latter will acquire a negative electrophoretic velocity which opposes the EOF velocity. The overall velocity can now be related to the electric field (E), the EOF mobility (μ L0P ), and the electrophoretic mobility (μ cp ). : o¡c¡uII = E(μ L0P + μ cp ) (2) The pH of the buffer affects the ionization of the analyte, which affects its effective electrophoretic mobility μ cp . For instance, glutamine in its zwitterion form (abbreviated as HA) has zero net charge and thus its electrophoretic mobility is zero, while glutamine in its anionic form (A - ) has a net negative charge and has an electrophoretic mobility μ ìon . Both forms of glutamine exist to a significant extent when the pH is within 2 units of the pKa of the ammonium group (one can confirm through the Henderson-Hasselbalch equation that at 2 pH units above the pKa, the ratio of anionic glutamine to zwitterionic glutamine is 100:1). Figure 2: Second ionization of glutamine occurs at the amino group Since only the anionic form contributes to the analyte’s effective electrophoretic mobility, then μ cp must be equal to the fraction of the anionic form of analyte multiplied by the anion’s electrophoretic mobility μ ìon . μ cp = μ ìon _ |A - ] |EA] + |A - ] _ (S) Procedure NOTE: THE PROCEDURES MUST BE FOLLOWED CLOSELY IN ORDER TO COMPLETE THE EXPERIMENT IN THE ALLOTTED TIME. In the SIF (room B354), begin setting up the CE instrumentation (Part A of the Appendix). The table below shows the contents of each vial that will be in the vial tray. Vial Solution 1 1M NaOH 2 Methanol 3 Deionized water 4 20mM pH 9.30 borate buffer 5 20mM pH 9.30 borate buffer 6 Deionized water 7 Solution 1 (blank) 8 Solution 2 9 Solution 3 10 Solution 4 11 Solution 5 12 Solution 6 13 Solution 7 (unknown) 20 Waste (empty vial) Table 1: CE Vial Contents Use separate pipettes to fill up the vials containing sodium hydroxide, deionized water, and buffer. Make sure vial 20 (waste vial) is empty. Then, load these vials (vials 1, 3, 4, 5, 6, 20) into the vial tray. Set up the instrument method described in Part B of the Appendix. Note that the method “CHEM3XX_CE.M” should have all the correct parameters in instrument method, so you only need to make sure it has not been altered. Use the Single Sample method described in Part C of the Appendix to run the deionized water sample. The data from this run is not analytically useful, but the run serves to ensure the instrument is flushed and prepared for your blank run (solution 1). The deionized water run will take approximately 15 minutes. During this time you will prepare your standards and unknown. Prepare 0.00375 g/mL asparagine and 0.0001 g/mL caffeine in the same 100 mL volumetric flask. Prepare 0.0075 g/mL glutamine solution in a separate 100 mL volumetric flask to the highest level of accuracy. The best way to accomplish this is to weigh by difference the solid glutamine and dissolve it in ~75 mL deionized water in a beaker. Use the sonicator to ensure complete dissolution and quantitatively transfer the solution to the volumetric flask before making to the mark. Prepare the seven solutions indicated below in 25 mL volumetric flasks. Dilute to 25 mL with deionized water. For the unknown solution (solution 7), it is recommended to dissolve the unknown in ~15 mL deionized water to allow room for the 5 mL of asparagine/caffeine solution. Solution asparagine/caffeine standard (mL) glutamine standard (mL) unknown (g) 1 (blank) 5 0 0 2 5 1 0 3 5 2 0 4 5 3 0 5 5 4 0 6 5 5 0 7 5 0 x Table 2: Preparation of standards and unknown Bring Solutions 1 to 7 to the SIF. Use a 1 mL syringe to inject Solution 1 through a 0.45 μm Durapore membrane filter into a capillary vial (Vial 7). Ideally, the vial should be about 7/8 full (around 0.6 mL). Load Vial 7 into the vial tray. In Sample Info (see Part C of Appendix), change the Vial Location to Vial 7 and rename the sample. Use Single Sample to run your first standard. While the sample is running, finish injecting your other samples into their respective capillary vials (see Table 1) using the same injection technique described for Solution 1. That is, you will inject Solution 2 through the filter into Vial 8, and so on. To avoid cross-contamination, when injecting a new sample, flush the filter unit at least twice with your new sample before filling the vial: simply inject 1 mL of the new solution through the filter into a waste beaker. As soon as a Single Sample run finishes, load the next sample vial into the vial tray, adjust the parameters in Sample Info, and run the sample. Once all the standards have been measured and you are in the process of running the unknown sample, check the electropherograms for consistency and confirm that you can plot a linear calibration curve from the data. Print out the overlay of the integration results for the standard solutions and unknown. See Part D of the Appendix. Calculations Determine the mass of the unknown glutamine in milligrams and report it on the first page of your report. Include the uncertainty and confidence level when reporting your result. In Your Report A full formal report is required for this experiment. Your report should include the following, but is not limited to: A description of the method of separation in capillary electrophoresis: use the learning objectives as a guideline for what you should discuss An explanation of why the internal standard is important to the method and how the internal standard was used in treating the data Include answers to the following questions in your report: 1. Explain the relative migration rates of the compounds used in this experiment. What would the relative migration rates be if pH 6.2 phosphate buffer is used? 2. Outline the appropriate conditions to determine the first ionization constant of glutamine (pKa of the carboxylic acid group). Hint: think about the buffer and capillary. References 1. Skoog, D.A., Holler J.F., Crouch S.R. Principle of Instrumental Analysis 6th Ed. Thompson Brooks: Belmont, 2007, Chapter 30. 2. Harris, D.C. Quantitative Chemical Analysis 7th Ed.W.H. Freeman and Co: New York, 2007, Chapter 26. 3. Solow, M. Weak Acid pKa Determination Using Capillary Zone Electrophoresis. J. Chem. Ed. 2006, 83 (8), p 1194-1195. Appendix A. Setup Turn on the computer and log in. Turn on the power button on the lower left of the Agilent Technology 7100 CE Instrument. In the Start Menu, open the program “Instrument 1 Online”. You will be greeted with this screen. Figure 3: Method and Run Control - Instrument Control Load the CHEM3XX_CE.M method. Note the file extension must be .M. In the lower left corner, you will see that you are in the Method and Run Control mode. The Run Method Task (single vial icon in top left corner) should be selected. Under the Instrument Control tab, you will have access to most of the commands related to initializing the instrument (On/Off), flushing the capillary, and running a single sample; once a sample is running, the sample identity is displayed at the bottom (circled in Figure 3), and the time remaining can be monitored. Turn on the UV lamp by clicking on the power icon within the DAD section. The inlet and outlet vials (circled in Figure 3) displayed on the instrument control interface refer to the vials in which the two electrodes are introduced. To remove the inlet (or outlet) vial from the instrument, the slot in the vial tray corresponding to the vial number must be empty and there must be another vial available for the electrode to dip into. Right click on the picture of the inlet vial and select “Set Inlet Vial”. A pop-up will then allow you to choose another vial as the inlet vial. For example, by replacing Vial 3 with Vial 2 as the inlet vial, Vial 3 will now be in the vial tray while the electrode will be dipped in Vial 2. The outlet vial can be switched in the same manner. Right clicking on the pressure gauge (circled in Figure 3) gives options for injection and flushing. The flushing option is useful towards the end of the experiment. Before flushing the capillary, make sure the outlet vial is set to Vial 20 (waste) and the inlet vial is set to the solution that you wish to flush the capillary with. To begin flushing, right click on the pressure gauge and select “Flush.” A pop-up will allow you to set the flushing time. B. Instrument Method Under the Instrument tab, click “Set up Instrument Method.” Under the DAD tab, make sure all the settings are the same as that shown in Figure 4. The must haves are: Signal: 210 nm with a bandwidth of 5 nm Stoptime: “As CE” Spectrum Range from: 190.0 nm to 400.0 nm UV Lamp required Figure 4: Setup Method – DAD Under the CE tab, the parameters on the left panel are: Inlet Home: 04 Buffer Outlet Home: 05 Buffer Cassette Temperature: 20.0 °C Enable High Voltage Voltage: 25.0 KV Current: 300 μA Power: 6.0 W Low Current alarm limit: Off Stoptime: 5:00 min Posttime: Off Under the CE tab, the right panel displays each step in a Single Sample run. Each run will start with preconditioning which consists of flushing the capillary for 4 minutes with buffer. This step is represented by a box under Preconditioning (see Figure 5). Steps can be created or removed using the “Insert” and “Delete” buttons. The parameters of each step can be set: the inlet, outlet, and duration can then be specified. For the injection step, the drop down menu on the left should display Apply Pressure; additional parameters include applying a pressure of 50 mbar for 5 s; the inlet is Injection Vial and the outlet is Outlet Home Vial. Following injection is postconditioning, which consists of two steps: the capillary is flushed with NaOH for 3 minutes, and then with deionized water for 3 minutes. Figure 5: Preconditioning, Injection, and Postconditioning Steps C. Single Sample Under the Run Control tab, click Sample Info. Fill out the file name and type CHEM3XX_CE as the subdirectory for the file to be saved. Then fill out the sample name and enter the vial location in Location. Figure 6 below shows the Sample Info for a deionized water run (vial location 6). To run the sample, either click Run Method or on the main screen click Single Sample. Figure 6: Single Sample - Sample Info To use the single sample command for other samples, simply change the sample name and specify the vial location. D. Data Analysis During the run, Instrument 1 offline can be opened by clicking on the Data Analysis tab near the bottom left. Under the File tab, click Load Signal. On the right of the Load Signal window, the CHEM3XX_CE folder where your files are saved can be accessed through the path: C:\CHEM32\1\DATA\CHEM3XX_CE. On the left, the data files contained in the selected folder are displayed. Click on Signal Details (near the bottom right of the Load Signal window); make sure that the Signal Description only contains the signal at 210nm. To remove other signals from Signal Description, select the entire row corresponding to the signal by clicking on the left margin of the row, and click Delete Row. To add a signal to Signal Description, select the desired signal from the drop down menu under Available Signals, and click Add to Method. In the Load Signal window, then select your data file from the left and click OK to load. If multiple signals are being displayed (for example the current, voltage, or power signals) then go back to File -> Load Signal -> Signal Details, delete all signals and reload the file. All available signals are displayed. Once again, go to File -> Load Signal -> Signal Details and add only the 210 nm signal to the method. Upon reloading the file, only the 210 nm signal will be displayed. Figure 7: Load Signal - Signal Details When all the electropherograms are overlaid, the automatic integration takes only a few seconds to integrate all the peaks. Once the signals for the blank, standard solutions, and unknown have been loaded, check the boxes next to all the runs as shown in Figure 8. Figure 8: Selecting Runs to Overlay Finally, in the area below “Sample Name”, double-click on the blank run. A pop-up will display a warning that too many signals are selected for overlay and will ask if you want to continue; click Yes to continue. All the selected signals will now be stacked one on top of the other as seen in Figure 9. Figure 9: Overlay of Signals Under the Integration tab, click the peak sign that says “Auto Integrate.” If necessary, use the manual integration tools (circled in Figure 9) to redraw or remove peaks. In File, go to Print, and click Integration Results. On the print-out, there will be no spectrum but only the tabulated integration results of each signal. Save your integration results by printing them to a PDF file, locating the file in the CHEM3XX_CE folder. Be cautious in interpreting the integration results; they are not always printed in an intuitive order. Print the electropherogram for one calibration solution and for the unknown. An overlay of these is acceptable. Before closing the program, the capillary needs to be flushed with methanol for 5 minutes followed by 5 minutes of deionized water. Note that methanol is volatile and you may have to refill Vial 2 using the HPLC grade methanol in the SIF. After the flush, set the inlet vial as Vial 3 and the outlet vial as Vial 6. Close Instrument 1 Offline, then close Instrument 1 Online, and finally shut off the instrument power. 65 A-08 Determination of Cr 3+ by Chemiluminometry LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Briefly describe the process of chemiluminescence Identify which of your reactants will be limiting the emitted light intensity Describe the advantages and disadvantages of the manual injection technique compared to the flow injection method Describe the principles and use of a photomultiplier tube (PMT) detector Recognize the safety concerns associated with using high-voltage power sources and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of Cr 3+ in your unknown using the calibration curve method and applying appropriate dilution factors Draw appropriate baselines for peak integration and height Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks, etc.) through practice BACKGROUND The oxidation of luminol by hydrogen peroxide in basic solution is a chemiluminescent reaction. The reaction is enhanced by chromium (III) but not by chromium (VI). Hence, chemiluminometry can distinguish between different chemical forms of chromium, this being a valuable feature in some biological applications. While the chromium is often referred to as a catalyst, the enhancement effect is not true catalysis in that the Cr (III) is consumed in the reaction. Many other metals enhance the oxidation, but interference by these species can be suppressed by the addition of ethylenediamine tetraacetic acid (EDTA). The inactive EDTA complexes form rapidly with the interfering metals, but only slowly with chromium. While your unknown does not deliberately contain metals other than chromium, this masking technique will prevent trace metal ions present in the deionized water from interfering with the analysis. Figure 1: Oxidation of luminol in basic solution Reaction times are short under the conditions of this experiment, and light is emitted for only a few seconds. Therefore the light must be measured sensitive to such factors as reagent concentrations, temperatur technique is critical, particularly during injection. The analysis will be done using both manual injection and flow injection analysis (FIA) techniques. In the former case optimal signal noise ratios are obtained by injec while in the latter case the S/N ratio is improved by injecting peroxide into a mixture of luminol and Cr (III). The reason for the difference is not obvious, but is probably related to the of the reaction and the very different rates of injection PROCEDURE Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few mL of water and add the washings to the flask. Dilute to the mark with water. Mix well. Using the 5.00E-06 g/mL chromium (III) stock s calibration solutions. Start with zero concentration (blank) and do not exceed 6.00E chromium for the most concentrated solution. Prepare a further five unknown. Ensure each solution is well mixed. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5, and 10 mL volume and 50mL volumetric flasks are provided. PART I: Analysis of Chromium by Manual Injection The manual chemiluminometer consists of a cell compartment equipped with a septum through which the sample is injected by syringe into a 1 cm optical cell. The emitted light is detected with a photomultiplier tube (PMT). The signal is converted to a voltage and amplified, then is digitized, stored, and processed by computer. Power on the computer and then open the Acquisition for Windows (AFW) program. Use the mouse to activate the buttons of the following tool bar. WARNING: The high voltage power supplies used are capable of producing lethal currents/voltage. Making of electrical connections must only be done with HV supply OFF. 66 : Oxidation of luminol in basic solution Reaction times are short under the conditions of this experiment, and light is emitted for only a few seconds. Therefore the light must be measured as the reactants are mixed. The reaction is sensitive to such factors as reagent concentrations, temperature, and mixing. Reproducible technique is critical, particularly during injection. The analysis will be done using both manual injection and flow injection analysis (FIA) techniques. In the former case optimal signal noise ratios are obtained by injecting the Cr (III) samples into a mixture of peroxide and luminol, while in the latter case the S/N ratio is improved by injecting peroxide into a mixture of luminol and Cr (III). The reason for the difference is not obvious, but is probably related to the of the reaction and the very different rates of injection used in the two techniques. Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few mL of water and add the washings to the flask. Dilute to the mark with water. Mix well. 06 g/mL chromium (III) stock solution, prepare at least five (preferably six) calibration solutions. Start with zero concentration (blank) and do not exceed 6.00E chromium for the most concentrated solution. Prepare a further five-fold dilution of your ution is well mixed. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5, and 10 mL volume and 50mL volumetric flasks are provided. PART I: Analysis of Chromium by Manual Injection The manual chemiluminometer consists of a cell compartment equipped with a septum through which the sample is injected by syringe into a 1 cm optical cell. The emitted light is detected with a photomultiplier tube (PMT). The signal is converted to a voltage and amplified, then is digitized, stored, and processed by computer. computer and then open the Acquisition for Windows (AFW) program. Use the mouse to activate the buttons of the following tool bar. The high voltage power supplies used are capable of producing lethal currents/voltage. Making of electrical connections must only be done with HV Reaction times are short under the conditions of this experiment, and light is emitted for only a the reactants are mixed. The reaction is e, and mixing. Reproducible technique is critical, particularly during injection. The analysis will be done using both manual injection and flow injection analysis (FIA) techniques. In the former case optimal signal-to- ting the Cr (III) samples into a mixture of peroxide and luminol, while in the latter case the S/N ratio is improved by injecting peroxide into a mixture of luminol and Cr (III). The reason for the difference is not obvious, but is probably related to the kinetics used in the two techniques. Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few mL of water and add the washings to the flask. Dilute to the mark with water. Mix well. olution, prepare at least five (preferably six) calibration solutions. Start with zero concentration (blank) and do not exceed 6.00E-07 g/mL fold dilution of your ution is well mixed. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5, and 10 mL volume The manual chemiluminometer consists of a cell compartment equipped with a septum through which the sample is injected by syringe into a 1 cm optical cell. The emitted light is detected with a photomultiplier tube (PMT). The signal is converted to a voltage and amplified, then is computer and then open the Acquisition for Windows (AFW) program. Use the The high voltage power supplies used are capable of producing lethal currents/voltage. Making of electrical connections must only be done with HV power 67 Click File then New to open a new file. Click Acquire, then Configure Run and set the following run parameters: Append to Current File √ (acquires data for several runs into one file) Rate 120.00 Additional Run Time 0.10 (entered through the keyboard) Temporary File Enable √ Click SAVE then OK. It should not be necessary to reset the configuration for the rest of the experiment. Power on the chemiluminescence amplifier and set the selector switch to MAN to monitor the photomultiplier tube of the manual apparatus. Turn on the magnetic stirrer. Five duplicate manual injections will be made for each standard solution and the unknown. Use the following procedure to charge the cell with peroxide and luminol solutions, and make test injections with the most concentrated chromium standard solution. • Turn on the high-voltage dc power supply with the button marked ‘MAINS’. Adjust the voltage to ~700 volts. Before proceeding, check that the ‘Output ON/OFF’ button of the supply is OFF (ie. there should be no “-ve” sign before the displayed voltage setting and the HV ON light should not be lit.) Remove the cell compartment cover. Flush the cell with water, then empty by aspiration. Add 1 mL of 0.0002 M luminol solution and 0.5 mL of 0.12 M hydrogen peroxide solution. Replace the cell compartment cover! The PMT can be damaged by exposure to room light. • Switch the Output of the power supply to ON so that -700 volts is applied to the PMT cathode. A “-ve” sign should appear before the displayed voltage. • To check that the baseline voltage is ~0.5 to 1.0 V, click “Acquire”, then “Manual Zero”. (The system records positive voltages only, and the offset ensures that the baseline will not drift to a negative value.) Close the “Manual Zero” window. • Click and enter the relevant information into the Header Labels window. Click SAVE and OK. • Fill a syringe with 5.00E-07 g/mL chromium solution (your most concentrated standard solution). Adjust the volume to 0.4 mL. Insert the syringe into the injection port, click OK 68 to initiate acquisition, and inject the solution after 1 to 2 seconds. NOTE! Reproducible injection technique is important! The injection itself should be as rapid as possible. • Switch the Output of the power supply to OFF, remove the cover of the cell compartment, and empty the cell by aspiration. Rinse the cell, then recharge with peroxide and luminol. • If the peak height for the first injection (using the most concentrated standard solution) is above 9 volts, reduce the PMT high voltage and repeat the injection. If the peak is less than 7 volts, increase the high voltage (1000 volts maximum!). Once the voltage has been set, do not change it for the rest of the analysis by manual injection. Click to initiate the acquisition routine for the next run. Acquire data for 5 injections of each standard solution, the diluted unknown, and the blank. Save the file. • If the software should happen to crash, advise your TA and/or instructor before attempting to restart the software. Processing the Data: Although the peaks can be identified automatically by computer (…try it - just push the button), the selected baselines will often be unsatisfactory. It is difficult to select a single set of criteria that will adequately define the wide range of peak shapes common in this experiment. It is likely that each displayed peak should be individually rescaled and processed manually. To rescale, hold down the left button of the mouse, and drag the cursor diagonally to draw a rectangle around the area to be displayed. Release the button. Click the button. Position the cursor at the start of the base of the peak, hold down the left key of the mouse, and draw the baseline. Release the button. Use the scroll bar to scan the data to the next peak. Draw a baseline for each peak on the displayed file. Turn off the button and click to reset the original display. Click to print the peaks. Click to display the data table, and to print the table. From the tabulated values of peak heights and areas, calculate the concentration of chromium (g/mL) in your original unknown solution as received in the vial. (Note: Inspect the data carefully before deciding upon the best method of calculation.) PART II: Assay of Chromium by FIA In FIA systems, sample or reagent "plugs" of 20 to 150µL volume are sequentially transferred from an injection loop by a carrier flow. The plugs are then taken through narrow-bore tubing to a flow-through cell. During delivery the plugs may undergo reaction with reagents in the carrier stream, or may combine with separate reagent streams. Various analytical instruments 69 (spectrometers, fluorometers, potentiometers, conductivity bridges etc.) can serve as detectors to monitor the cell. The FIA system used in this experiment consists of the following components. • a peristaltic pump that maintains constant flow rates of solutions through four narrow bore tubes, • an 8-port automatic valve that allows for multiple injections of 25µL plugs of hydrogen peroxide from an injection loop into a carrier stream of water, • a spiral flow-through cell in which the analyte is mixed with the reagents, • a photomultiplier tube (and amplifier) that monitors the light emitted from the cell, • a computer, equipped with an analog-to-digital converter, that acquires and processes the amplified signal from the photomultiplier tube. The system status, in each of the possible valve positions, is as follows: LOAD POSITION: • The injection loop is filled with peroxide. Excess is pumped to waste. • Sample is mixed with luminol. The mixture is merged with the carrier stream (water) at the cell. INJECT POSITION: • The water stream is redirected through the injection loop, carrying the peroxide plug to the cell where it reacts with the mixture of luminol and sample. • H 2 O 2 stock solution is pumped directly to waste. Initial settings for Alitea C- 4V pump: Obtain help in fitting the four silicone tubes around the roller drum of the Alitea peristaltic pump, then make the following settings on the pump: POWER ON/OFF ON CLOCKWISE/COUNTERCLOCKWISE CW SPEED 500 LUMINOL SAMPLE H2O2 WATER INJ. LOOP PUMP WASTE WASTE VALVE LOAD POSITION DETECTOR (PMT) 0.8ml/min 0.8ml/min 0.8ml/min 1.3ml/min LUMINOL SAMPLE H2O2 WATER INJ. LOOP PUMP WASTE WASTE VALVE INJECT POSITION DETECTOR (PMT) hv 0.8ml/min 0.8ml/min 0.8ml/min 1.3ml/min 70 START/STOP STOP Initial settings for 8-port valve controller: POWER I/O I (i.e. ON) T1 (load time) 6.0 (~30 SECONDS) T2 (inject time) 2.0 (~10 SECONDS) POSITION A CONTROL AUTO START/RESET RESET Computer and amplifier: Set the chemiluminescence amplifier to monitor the PMT of the FIA system. A single acquisition file of 30 minutes should be sufficient to acquire the data for all solutions. Click Acquire, then Configure Run and set the following run parameters: Append to Current File disable Rate 30.00 Run Time 30 Temporary File Enable √ • Dilute 30 mL of 0.12 M hydrogen peroxide to about 100 mL with deionized water. Immerse the inlet ends of the appropriate silicone tubes into three 125 mL Erlenmeyer flasks containing ∼100 mL respectively of water, 0.0002 M luminol, and diluted hydrogen peroxide. • Insert the end of the tube labeled "sample" into the volumetric flask containing the most concentrated Cr(III) standard. Insert the two outlet tubes into a waste container. • Press "START" on the pump. To ensure that the system is pumping satisfactorily, momentarily lift each tube from the solution. An air bubble should pump through the tube. Switch the valve controller to "RESET". The valve will rotate repeatedly through load/inject cycles. (The green LED indicates when the valve is in the "inject" position). Switch the valve to "START". • Switch on the dc power supply so that about 600 volts is applied to the PMT cathode. Click to start the acquisition of data. Adjust the dc voltage until the displayed peaks are about 90% of full scale. Do not change this setting for the rest of the experiment. Once 5 satisfactory peaks have been recorded, quickly and smoothly remove the "sample" tube and insert it into the volumetric flask containing the next standard solution. Record five satisfactory peaks for each standard solution, the blank, and the unknown. 71 • When finished, insert all inlet tubes into a flask of deionized water and flush the system thoroughly. Obtain help in releasing the silicone tubes from the pump and shutting down the system. Process the peak data as before. From the tabulated values of peak height and peak area, calculate the concentration in g/mL of chromium in your original unknown solution as received in the vial. IN YOUR REPORT Report both the [Cr] determined by FIA and by manual injection technique on the front cover of your report. Compare the FIA results with those using the manual injection. In comparing your results, make sure to use the appropriate statistical tests to determine whether the results agree. Using the data from the five blank runs, calculate the signal detection limit and the minimum detectable concentration for both the manual and FIA techniques. Ensure that the units of these two values are given correctly. BACKGROUND READING 1. Hanson, E.H.; Ruzicka, J. J. Chem. Ed. 1979, 156(10), 677. (Hanson and Ruzicka are the co-inventors of the FIA technique). 2. Ruzicka, J.; Hansen, E.H. Flow Injection Analysis, Wiley: New York, 1988. (QD79.I5 R89 1988 in IKBLC) 3. Wade, A.P.; Shiundu, P.M.; Wentzell, P.D. Anal. Chim. Acta 1990, 237, 361. 4. Ruzicka, J.; Hansen, E.H. Anal. Chem. 2000, 72(5), 212A. 5. Seitz, W.R.; Suydam, W.W.; Hercules, D.M. Anal. Chem. 1972, 44, 957. 72 A-09 Determination of Calcium in the Presence of Aluminum by Atomic Absorption Spectrometry LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the processes occurring between sample introduction and analyte atomization in the flame Describe the effect of aluminum on the calcium signal strength and how addition of strontium can overcome the interference. Describe the advantages and disadvantages of the calibration curve method compared to the standard addition method Recognize the safety concerns associated with igniting, using and extinguishing an air-acetylene flame and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of Ca 2+ in your unknown using the calibration curve method and standard addition method applying appropriate dilution factors Explain the flame colour changes that occur upon introduction of different solutions including the variation in colour with height in the flame Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks, etc.) through substantial practice BACKGROUND Unlike the absorption and emission spectra of molecules, the absorption and emission spectra of atoms are characterized by very narrow bandwidth. From an analysis perspective, these narrow bandwidths both complicate the analysis by requiring precise instrumental control over wavelength and also improve the analysis by providing ‘built-in’ specificity: it is unlikely that any two atomic lines overlap. Instead of suffering spectral interferences, these analyses are more susceptible to chemical interference. In this experiment, we examine the specific interference of aluminum when trying to determine calcium, and demonstrate one particular technique for minimizing the effect of any aluminum present. To begin, we first need to think about one of the key steps in atomic spectrometry: production of the analyte in atomic (i.e. not molecular) form. The atomic absorption of calcium is normally measured by means of a reducing (fuel-rich) air-acetylene flame. In an oxidizing (oxidant-rich) flame calcium is partially converted to oxide and, there being fewer free atoms, the sensitivity is 73 diminished. The measurement is subject to chemical interference, as opposed to spectral interference, from several cations and anions. For example, aluminum depresses the calcium signal because of the formation of the refractory compound calcium aluminate which passes through the flame as a solid without dissociation into atoms. This interference can be substantially removed by the addition of a strontium salt as a so-called releasing agent. In sufficient quantity the strontium ties up the aluminum as refractory strontium aluminate, and the calcium then exists in the flame mainly in the atomic state. Strontium also controls the ionization of calcium in the flame. A correction must be made for the calcium usually found as an impurity in strontium salts. Residual interference can be compensated by the technique of standard addition, whereby measurements are conducted on a series of solutions, each of which is prepared from a fixed volume of unknown solution together with one of a range of volumes of standard solution. Provided the resulting plot of absorbance versus concentration of added standard is linear, the concentration of the unknown solution can be obtained by extrapolation to the concentration axis. The standard addition technique should be applied with discretion. It sometimes cannot cope alone with gross chemical interference (why not?), so should preferably be combined with the releasing technique. PROCEDURE Your unknown solution contains aluminum as well as calcium. Add all of your unknown solution to a 250 mL volumetric flask. Rinse the vial with a few millilitres of water and add the washings to the flask. Dilute to the mark with water. Mix well. The first runs will demonstrate the interference of aluminium and the releasing action of strontium: Part 1 – Qualitative Analysis Run 1 – Determining the signal strength with calcium alone: To a 50 mL volumetric flasks add 5.00 mL of a solution containing 4.00E-05 g/mL calcium. Dilute to the mark with water. Measure the calcium absorbance using water as a reference. Run 2 - Interference by aluminum: To a 50 mL flask add 5.00 mL of a solution containing 4.00E-05 g/mL calcium and 1 mL of 4.00E-04 g/mL aluminum, dilute to the mark. Put 1 mL of 4.00E-04 g/mL aluminum in a 50 mL flask dilute to the mark to use as a blank. Measure the absorbance of calcium using the blank solution (not water) as the reference. WARNING: Ignition, use and extinguishing the air-acetylene flame is a potential-explosive action. Strict protocols are given in the lab manual and must be followed. As always, lab coat and eye protection must be worn. With some AA instruments, the flame is large and poorly-shielded. Care should be taken to avoid accidental contact with the flame or nearby hot surfaces. Atomic emission of some elements produces UV light. The instrument operator should avoid staring at the flame. 74 Run 3 - Releasing action of strontium: Proceed as in Run 2 but add 5 mL of 1% strontium chloride solution to each flask before diluting. Use blank solution as the reference. Refer to the appendix and use the Perkin-Elmer 305A atomic absorption spectrophotometer to measure the absorbance of calcium in an air-acetylene flame for each of the calcium-containing solutions. Record the absorbance for each run. Part 2 – Quantitative analysis Clean the 50 mL flasks and prepare the following three sets of solutions. The measured absorbances will be used to determine calcium in the unknown sample. In run 4 calcium will be determined using the releasing technique alone, in run 5 it will be determined using the combined releasing and standard addition techniques, and in run 6 it will be determined using the standard addition technique alone. Run 4 - releasing technique: Using the 4.00E-05 g/mL calcium stock solution, prepare at least five (preferably six) calibration solutions. Start with zero concentration (blank) and do not exceed 4.5E-06 g/mL calcium for the most concentrated solution. Prepare a further five-fold dilution of your unknown. In each volumetric flask, also add 5 mL of 1% (w/w) strontium chloride solution. Do not add aluminum solution. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 50mL volumetric flasks are provided. Ensure each solution is well mixed. Measure the absorbance of calcium using the blank solution as the reference. Plot your calibration curve and by interpolation determine the concentration in g/mL of calcium in your original unknown solution. Run 5 - combined releasing and standard addition techniques: To each of six 50 mL volumetric flasks add 10.00 mL of diluted unknown solution. Add 4.00E- 5 g/mL calcium stock solution sufficient to have solutions with added calcium in the range of zero to 2.00E-4 g. To each flask add 5 mL of 1% strontium chloride, but no aluminum solution. Prepare a blank solution by diluting 5 mL of 1% strontium chloride solution to 50 mL. Measure the absorbance of calcium using your blank solution for the reference. Plot your standard addition line and by extrapolation determine the concentration in g/mL of calcium in your original unknown solution. Run 6 - standard addition technique: Finally, for comparison, apply the standard addition technique without the releasing technique as follows. Proceed as in the last run, but omit the addition of strontium chloride. Use deionized water as the reference. 75 IN YOUR REPORT Of the three determinations of unknown choose the best and report only one calcium concentration, in g/mL, on the first page of your lab report. Compare and discuss the results obtained with the different methods. In comparing your results, make sure to use the appropriate statistical tests to determine whether the results agree. Explain fully why the measured calcium absorbance differed between runs 1 and 2. Describe the role strontium played in determining your results for run 3. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6 th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 9. 2. Harris, D.A. Quantitative Chemical Analysis, 8 th ed.; W.H. Freeman: New York, 2010; chapters 21-1, 21-2 and 21-5. 76 APPENDIX I: Use of Perkin-Elmer 305A Spectrophotometer CAUTION: INCORRECT USE OF THE AIR-ACETYLENE BURNER COULD RESULT IN A “FLASH-BACK” EXPLOSION. Consult your TA before proceeding and note the following safety precautions: • The drain tubing loop must always be filled with water • The air supply must always be turned on before the acetylene supply is turned on • The air supply must always be turned OFF after the acetylene supply is turned off Operating Instructions: 1) Ensure that the AIR/ACETYLENE burner is installed. 2) Ensure that the loop in the plastic drain tubing is filled with water and the end of the tube is inserted in the drain reservoir (on the floor). 3) Turn SOURCE fully counterclockwise. Push POWER on to power the instrument and the exhaust fan. 4) Lighting the flame: Ensure that the air compressor is switched on (ask for help if it is not). Switch on AIR using the black valve. Monitor the pressure gauge and set the air pressure to 30 psi with the silver knob. Monitor the flow meter and set the air flow rate to 9 (middle of ball). Open the acetylene cylinder and if necessary, set the supply pressure to 12 – 15 psi. Open the FUEL SHUT OFF valve. Monitor the pressure gauge and adjust fuel pressure to 8 psi with the silver knob. Monitor the flow meter and set the fuel flow rate to 9 (middle of ball). Light the flame from below with the ignitor (wear leather gloves). Allow the burner chamber to warm-up for 10 minutes. 77 5) Instrument settings: Monitor the ammeter (current meter) inside the lamp compartment and use SOURCE to set the lamp operating current at 10 mA. Set FILTER to OFF (button not lit) Set PHASE to “NORMAL”. Turn EM CHOPPER to “OFF”. Set DAMPING at “2”. Set SLIT at “4”. Set MODE to “ABS”. Set RANGE to “VIS”. Set the WAVELENGTH initially at 211.3 (this sets λ at about 422.6 nm), then make a fine adjustment (up or down) until the ENERGY meter indicates a maximum reading. Adjust the GAIN until the needle of the ENERGY meter is in the dark red zone. 6) Reading the absorbance: (NOTE – aspirate deionized water into the burner between measurements.) Turn the EXPANSION knob fully counterclockwise. Switch the DIRECT/NULL knob to “NULL”. Use the absorbance knob to zero the digital readout of ABSORBANCE. Aspirate the appropriate reference solution into the burner. Use the ZERO knob to set the meter needle to zero. While aspirating the test sample into the burner, use the absorbance knob to reset the meter needle at zero. Record the absorbance reading from the digital meter. After reading the absorbances of the solutions in each set, aspirate the blank solution and re-check the zero setting. For each set of solutions plot the absorbance readings as they are taken. 7) Shut-down procedure: (OBTAIN ASSISTANCE) Turn the FUEL SHUT OFF lever down. Close the valve on the acetylene cylinder. Open the FUEL SHUT OFF lever to bleed the lines. Once the pressure gauge reads zero, close the fuel shut off lever. Turn the black gas control lever from “AIR” to “OFF”. Turn the POWER to “OFF” Turn the SOURCE and GAIN knobs fully counterclockwise. Turn off the air compressor only if no-one else is using any the flame AA instruments. 78 A-11 Determination of Copper by ICP-MS – Isotope Dilution MS LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the way in which the argon plasma is generated and sustained Describe the processes that occur when sample is introduced into the plasma Describe the components of the vacuum system that allow for sample introduction at atmospheric pressure and detection under high vacuum. Explain the advantages and disadvantages of the ratio method compared to the isotope dilution method. Recognize the safety concerns associated with nitric acid use and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of Cu in your unknown using the ratio method and isotope dilution method applying appropriate dilution factors Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks, etc.) through practice BACKGROUND Inductively-coupled plasma mass spectrometry (ICP-MS) is a highly-sensitive analytical method suitable for the determination of trace levels of multiple elements or isotopes. The combination of high-sensitivity and rapid determination of multiple elements makes the ICP-MS a valuable instrument in analytical labs. They’re also valuable to the manufacturers; a new ICP-MS costs ~$250,000! In the instrument, the sample is nebulized into an argon plasma at a temperature of ~6000°C. Analyte isotopes are converted to charged ions – typically M + – and are introduced through a partially-evacuated interface into a mass spectrometer operating under high vacuum. Ion intensity (counts/sec) is output as a function of mass-to-charge ratio, m/z (or AMU for ions with a charge of +1). Analyte concentrations are typically in the sub-ppb to low-ppm range. At these concentrations good experimental technique is critical. Background corrections are particularly important, as are the methods of treating uncertainties in the instrumental measurements. Many of these uncertainties in ICP-MS can be minimized through use of an elegant technique called “isotope dilution mass spectrometry” (IDMS). 79 Most elements from natural sources exist as mixtures of isotopes having well-defined relative abundances (see appended Table 1), although a few notable exceptions such as Pb have isotopic abundances that vary depending upon the source of the element. (Isotopes of Pb are end- products of the radioactive decay of uranium and thorium.) In IDMS, a “spike” enriched in one of the isotopes of the analyte element is added to the unknown sample. The mass spectrometer measures the enriched isotope and one other isotope of the analyte element. The concentration of the element present in the original unknown is calculated from the mass spectrum, and the published values of the isotopic compositions of the original analyte (Table 1) and the “spike”. The “spike” serves both as an ideal internal standard (why is it ideal?), and as an added standard of the analyte. Copper from natural sources contains two isotopes, 63 Cu and 65 Cu. In the IDMS analysis of copper, a mass spectrum is recorded of a solution containing the Cu analyte (natural isotopic abundance) to which has been added an aliquot of copper standard enriched in 65 Cu. Schematic representations of the mass spectra of the unknown, the spike solution, and the mixture are shown in Fig. 1. The fractional isotopic abundances ( ) u 63 Cu f and ( ) u 65 Cu f of the Cu in the original unknown are given in table 1. The fractional abundances ( ) s 63 Cu f and ( ) s 65 Cu f of the copper in the 65 Cu-enriched spiking solution are furnished by the supplier or can be calculated from your data. (Note that all isotopic abundances f are in units of mole fraction, and not weight fraction; these differ since the mass of any one isotope of copper is distinct from the copper atomic mass found on the periodic table.) i o n i n t e n s i t y mass number FIG. 1a: Unknown Cu (natural relative isotopic abundance). i o n i n t e n s i t y mass number FIG. 1b: “Spike” solution isotopically enriched in 65 Cu. mass number i o n i n t e n s i t y FIG. 1c: Unknown Cu spiked with isotopically enriched 65 Cu. 80 The sensitivity of the instrument towards 63 Cu and 65 Cu should be expected to be similar but not equal. Variation in the efficiency of the plasma in producing the ionized atoms, as well as differences in quadrupole and detector response mean that the sensitivities are likely slightly different. The counting efficiency k (≈1) evaluates this experimentally from the mass spectrum of the copper in the original, natural source, unknown. ( ) ( ) ( ) ( ) u 65 u 63 u 65 u 63 Cu u Cu u Cu Cu f Cu moles f Cu moles k counts counts × = (1) and therefore: ( ) ( ) ( ) ( ) u 65 u 63 u 65 u 63 Cu Cu Cu Cu f f counts counts k = (2) where ( ) u 63 Cu f and ( ) u 65 Cu f are the abundances listed in table 1. k is used in later calculations as a normalizing factor. From Fig.1c the count ratio R of 63 Cu to 65 Cu for the spiked unknown solution is: ( ) ( ) ( ) ( ) ( ) ( ) s 65 u 65 s 63 u 63 s u 65 s u 63 Cu s Cu u Cu s Cu u Cu Cu f Cu moles f Cu moles f Cu moles f Cu moles k counts counts R + + × = = + + (3) where the amounts in moles referred to correspond to amounts in the 50ml flask. Solving for the amount of copper (sum of isotopes) in the unknown gives: ( ) ( ) ( ) ( ) u u s s Cu Cu Cu Cu s u f f k R f k R f Cu moles Cu moles 63 65 65 63 − − = (4) In addition to the isotope dilution method above, analysis by ICP-MS can be accomplished by measurement of an unknown and a standard much like most other instrumental methods. Here, the isotopic nature of the sample (natural abundance or enriched) must be the same for both unknown and standard. Concentration in the unknown is calculated from the ratio of the counts for a chosen isotope. This method is useful for multi-element analysis, by which as many as 81 different elements can be quantified from a single mass spectrum. In this experiment Cu will be analyzed by quantification against a known Cu standard of natural isotopic abundance and by IDMS. 81 NOTE: Students will do this experiment individually, not in pairs. Each student will receive a different unknown solution. PROCEDURE At the start of the laboratory period, check that the ELAN 6000 ICPMS is pumped down. If it is not, refer to the APPENDIX and get help from the TA to initiate the startup procedure. DO NOT ignite the plasma at this time. To five 50mL PMP volumetric flasks add the following volumes of unknown, a solution containing 5.00E-07g/mL Cu (natural isotopic abundance), and a solution containing 5E-07g/mL of Cu isotopically enriched in 65 Cu. (NOTE: record the standardized concentration of this latter solution!) Dilute to the mark with 1% EG HNO 3 . NOTE 1a: Plasticware rather than glassware must be used for these trace level analyses. Contaminants adhering to the surface of glass, even after thorough cleaning, give unacceptably high background levels of the analytes. The polymethyl-pentene (PMP) plasticware used in this experiment must be rinsed at the beginning of each period, first with a few mL of 3% AR nitric acid provided especially for cleaning – do NOT use the environmental grade acid for cleaning – and then several times with deionized water. The same sample of acid can be cycled between several pieces of plasticware. The AR nitric acid is not suitable for preparing your solutions! NOTE 1b: The solutions used in this experiment are very expensive and must not be contaminated. The ultrapure acid costs $500/liter, and the stock 10µg/mL 65 Cu spiking solution is $6/mL (or $600,000 per gram of 65 Cu). A small amount of the 65 Cu spiking solution will be provided in a plastic bottle from which aliquots can be pipetted directly using a dedicated micropipetter. Therefore it is NOT necessary to rinse the micropipetter with the spiking solution. Add 1% HNO 3 to the volumetric flasks from a minimum volume of acid contained in a clean 250mL PMP beaker. Never return excess to the bottle. Prepare the following solutions with a micropipetter using new disposable plastic tips. A plastic dropper is provided for diluting solutions to the mark with 1% HNO 3 . WARNING: Nitric acid solutions are corrosive and act as strong oxidizers. Avoid skin contact. Rinse immediately with cold water if it occurs. 82 Solution Vol. unknown (mL) Vol. 5E-07g/mL Cu (natural isotopic ratio) (mL)* Vol. 5E-07g/mL 65 Cu-enriched standard (mL)* Add 1% HNO 3 to dilute to (mL) 1 0 0 0 50.00 2 2.00 0 0 50.00 3 0 0 2.00 50.00 4 2.00 0 2.00 50.00 5 0 2.00 0 50.00 * NOTE: Record the exact concentration of the stock solution of 65 Cu-enriched standard indicated on the bottle. Both Cu stock solutions contain 1% nitric acid. Refer to the Appendix for operating instructions for the ELAN 6000 ICP-MS and obtain five sets of counts at mass numbers of 63 and 65 for each of the solutions. CALCULATIONS Quote all final results in units of ppb. 4. Use the data for solution 1 to determine background corrections for all calculations. 5. Use the data for solution 3 to check the purity of the 65 Cu-enriched spiking solution. Compare your calculated value to the supplier’s purity certificate posted in the lab. 6. Calculate k (Eq’n 2) from the mass spectral data for copper of natural isotopic abundance (solutions 2 or 5). 7. Calculate R (Eq’n 3), the count ratio for spiked unknown (solution 4). 8. Calculate the concentration of Cu in your original unknown by IDMS (Eq’n 4) using k, R, the concentration of the 65 Cu spiking solution and the published molar isotopic abundances of 63 Cu and 65 Cu in the unknown and spiking solutions. 9. Calculate the concentration of Cu in your original unknown by ratio. Use the count data for either 63 Cu or 65 Cu in solutions 2 and 5. Remember that solutions 2 and 5 both contain Cu of natural isotopic abundance. IN YOUR REPORT Report both your IDMS and ratio method result on the cover of your report. Use units of ppb. Justify your choice ( 63 Cu or 65 Cu) used in the calculation by ratio. Refer to Table 1 and explain why 63 Cu and 101 Ru are the isotopes recommended for analyses of Cu and Ru respectively by this second method. Compare this result with that calculated by IDMS. In comparing your results, make sure to use the appropriate statistical tests to determine whether the results agree. 83 BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6 th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 11 (especially 11C). 2. Platzner, I.T.; Habfast, K.; Walder, A.J.; Goetz, A. Modern Isotope Ratio Mass Spectrometry; Wiley: New York, 1997. (QD96.M3 P57 1997 in IKBLC ASRS) 84 APPENDIX: Operating Instructions for the ELAN 6000 ICP-MS Do not try to use this instrument without the help of a teaching assistant! It is left on at all times with the mass spectrometer pumped down and thermally-stabilized. Do not ignite the plasma until you have prepared your solutions. The plasma consumes argon at a rate of ~17 to 20 liters/min. The monitor display may appear in a number of configurations depending upon the status of the instrument. However the following toolbar will appear at the top of the display: Use only the buttons described on the following pages. Some of the other buttons are for tuning and optimizing the mass spectrometer section of the instrument. If used incorrectly, they will detune the instrument thereby affecting adversely your data. Click to open the instrument control window. If necessary, you may have to click the “Front Panel” tab to display the following window: 85 The displayed graphic includes the pumps, the mass spectrometer housing, the nebulizer, and the argon gas lines. Be sure to include in your report a short description of the function of these components as well as the torch, the RF load coil positioned around the nozzle of the torch, the interface between the torch and the mass spectrometer, and the mass spectrometer itself. The sample solutions will be pumped into the nebulizer at a rate of 0.8 to 0.9 mL/min using a peristaltic pump. The inlet tube to the nebulizer is labeled with black retaining tags, and the outlet tube is labeled with white tags. The outlet tube is slightly larger than the inlet tube to ensure that test solutions will not accumulate in the nebulizer housing. Insert the tube with the black tag into a 100mL beaker of 1% EG nitric acid. The tube with the white tag should drain into a beaker or flask. Set the pump at ~ -15.0 to give an optimal CCW flow rate of 0.8 to 0.9 mL/min. Acid should be pumped into the nebulizer housing whenever solutions are not being measured. When you are ready to make measurements, click “Start” under “Plasma” to initiate the ignition sequence (~1 minute). The plasma can be viewed through the window on the top right of the instrument. Allow the plasma to stabilize for about 15 minutes before making measurements. Recording the mass spectra: Several files are required for the operation of the ELAN 6000, and the choice of these files will depend upon the experiment. The files define the tuning and optimization of the instrument, the method of acquiring data, the output of reports, etc. A “Workspace” file saves and recovers all the files needed for a given experiment. Click File, Open Workspace, and open “Chem 3XX- 425 Quantification.wrk”. If a box indicating “No analytes to measure” is displayed, click OK. Click to open the method file which defines the measurement parameters. Click the “Processing” then “Timing” tabs, and check and/or set the following: “Sweeps/Reading” 10 “Readings/Replicate” 1 “Replicates” 5 Check that the two isotopes of copper, and no other isotopes, have been selected in the Analytes table. To remove isotopes from the list, click the numbered cell to the left of the isotope, then click “Edit”, “Cut Row” at the top of the window. To add isotopes to the list, right click on any cell in the “Analyte” column thereby displaying a periodic table. Single click on elements to select them, and double click to deselect them. The software defaults to selecting the most commonly used isotope for each element. However this IDMS analysis requires that both isotopes of Cu be measured. Hold down CTRL SHIFT, click on both isotopes, and click OK. The “Scan mode” will default to “Peak Hopping” in order that data will be measured at the center of the peak for each isotope and not between the peaks. Close the “Data Only Method” window. 86 Click then the “Manual” tab to open the Sample window. Click “Details” and enter “Cu” and your initials in the “Sample ID” box, and the sample description (“blank”, “unknown”, “spike” etc.) and your initials in the “Description” box. This information will be printed on the final report. Click to open the Realtime window in which the data will be displayed during the run. Click “Analytes” to confirm the two isotopes of Cu have been selected. If not, click “Add All”. Click OK. The box at the top left of the Realtime window should be set to “Numeric”, and not “Spectral” or “Signal”. Rinse the peristaltic pump inlet tube with deionized water, and then insert it into the flask containing your blank solution. Pump blank through the system for 2 minutes, and then click the “Analyze Sample” button to acquire data into the Realtime window. Do not use the “Analyze Blank” function when measuring the blank. Click to display the collected data. Print the raw data. Pump 1% environmental grade HNO 3 through the system between samples. Similarly collect data for all five solutions. Remember to make appropriate entries into the “Description” box in the Sample window before running each sample. Once all samples have been measured, rinse the plasticware thoroughly with deionized water, then with your remaining blank solution (1% nitric acid). Flush again with DI water. To turn off the plasma click the “Stop” button under “Plasma”. Do NOT turn off the vacuum or the argon supply! Use the peristaltic pump to flush the system with 1% nitric acid for 10 minutes, then stop the pump and release the silicone tubes from the drum. Table 1 is on the following page. 87 88 A-12 Determination of Manganese by Neutron Activation Analysis LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe how 55 Mn is activated to form 56 Mn Describe the radioactive decay of 56 Mn Describe how radioactive decay events are detected by the scintillation counter Describe how you will use statistical analysis of the radioactive decay from a tritium sample to assess whether the scintillation counter is producing expected results Recognize the safety concerns associated handling of radioactive samples and plan to use best practices On completion of the experiment and lab report, you should be able to: Correctly apply correction factors to your data to give a dataset that directly relates concentration and decay counts Correctly calculate the concentration of Mn in your unknown using a calibration curve applying appropriate dilution factors Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks, etc.) through practice BACKGROUND Chemical analysis by neutron activation exploits the radioactive properties of materials in order to determine their amount. Radioactive decay events are counted, and this count related to the amount of material present. Alas, there are complicating factors: 1) Although larger amounts of radioactive material will give more decay events, we must also consider that radioactive decay is a random process. If the number of decay events in a given period of time is measured several times, the number of decays will generally vary between measurements. To deal with this, careful application of statistical analysis is needed and this is described in detail later. 2) Not all materials are naturally radioactive, limiting the number of analytes which can be determined by counting decay events. One can widen the range of possible analytes by making them radioactive. This process is termed ‘activation’. In this experiment, you will activate your aqueous unknown sample of natural, non-radioactive manganese ( 55 Mn) into radioactive 56 Mn by bombarding it with neutrons. The radioactivity of the freshly-prepared 56 Mn sample is then measured via the Cerenkov effect, in which beta particles emitted by the 56 Mn generate pulses of light as they travel through a solvent solution. The light pulses are detected and counted with a scintillation counter. 89 Dealing with the random nature of radioactive decay: 56 Mn decays via the release of a beta particle and subsequent emission of gamma photon(s). Like other nuclear processes, the time-sequence of these releases is random in nature. In a series of equal time intervals, different numbers of photons are emitted. The frequency of occurrence of particular counts during the time intervals follows a Poisson distribution, P e x x x = −µ µ ! where P x is the probability of obtaining a count of x, and µ is the mean count after some number of measurements. Increasing the number of measurements will give µ increasingly closer to the ‘true value’. For any finite number of measurements, the best analytical data possible is a ‘best value’ for the mean count and some associated uncertainty. This ‘best value’ of decay counts will be used to quantify the amount of 56 Mn present. We must now consider how we will verify that a Poisson distribution is experimentally-achieved and how we will characterize the uncertainty in the mean count. We note that since x, but not necessarily µ, is an integer, we can represent measured data as a histogram and observe whether the histogram appears similar to a model discontinuous Poisson distribution. If the number of repeated measurements is small (<10), the histogram is assymetrical. However, if the number of repeated measurements is larger (>10), the shape of the histogram is symmetrical and resembles a Gaussian curve. Taking the standard deviation of this Gaussian curve allows us to define the uncertainty in our mean count. Whether or not we observe a Poisson distribution of the measured data depends on two factors: if the underlying process is random in nature and if the measurement method is not biased. We know that the radioactive decay of 56 Mn is a random process, but we should check that our measurement method is suitable. Is the counting efficiency of the instrument stable over some given period of time? Does the fluctuation in the decay count rate seem too variable? Too consistent? Any of these would point to instrumental bias in counting the decay events. To quantify the terms ‘too variable’ and ‘too consistent’ we use a chi-squared test (aside: ‘chi’ rhymes with ‘guy’). We make use of a fundamental property of the Poisson distribution model: that the average value and the variance (square of the standard deviation) are equal. That is, the standard deviation is given by µ 1/2⁄ . As long as we have a high µ the conformity of experimental observations to a Poisson distribution (taking into account the limited number of observations, N) can be tested by statistical means. We compare the average and the standard deviation of the measured data, but taking the ratio of the two to calculate the ‘coefficient of variation’: 1 2 ≈ x S x 90 For data which is perfectly Poissonian, the ratio should be exactly 1. If the ratio is much larger than one, the data are over-dispersed (more variable than expected), and if the ratio is much smaller than one, the data are under-dispersed (less variable than expected). We use this to test the goodness-of-fit of the data to a Poisson model. We calculate D, the index of dispersion: D N S x x = − ( ) 1 2 This is just N-1 times the coefficient of variation. As statisticians like to do, we hypothesize: 2 1 − ≈ N D χ Which says that under a Poisson model, the value D should follow the Chi-squared distribution for N-1 degrees of freedom. This Chi-squared distribution characterizes the variability we expect to see in D for a given number of measurements. If the data are perfectly Poissonian, the two terms should be equal. Given real experimental data, you are unlikely to find this equality. The question is then: How close do the index of dispersion and the Chi-squared distribution need to be to indicate that the data are likely Poissonian? First, we need to choose a confidence level. In keeping with the usual convention in analytical chemistry, we choose 95%. Then we compare our calculated value of D with critical values for Chi-squared. This is very similar to comparing a calculated t value against a tabulated t value in the Student t-test. However, in this case we use a so-called ‘two-sided’ test. That is, we are interested to know if our data are either over- dispersed or under-dispersed. This means that we compare our D value to both a lower critical value and an upper critical value. Values at the 95% confidence level are tabulated on the following page. If the calculated value of D lies between the lower and upper critical value, our hypothesis is true and we accept that our data fit a Poisson model. If D is either below the lower or above the upper value, the test shows that our data do not fit the Poisson model at the 95% confidence level (i.e. there is still a 5% chance the data actually is Poissonian). If your data do not appear Poissonian, faulty equipment or technique may be the problem. In this experiment, verification that the scintillation counter is unbiased is done by repeatedly measuring the decay of a tritium sample and calculating D. 91 Degrees of Freedom Critical Values for chi-square distribution at 95% CL (n-1) Lower Upper 15 6.262 27.488 16 6.908 28.845 17 7.564 30.191 18 8.231 31.526 19 8.907 32.852 20 9.591 34.170 21 10.283 35.479 22 10.982 36.781 23 11.689 38.076 24 12.401 39.364 25 13.120 40.646 26 13.884 41.923 27 14.573 43.195 28 15.308 44.461 29 16.047 45.722 30 16.791 46.979 31 17.539 48.232 32 18.291 49.480 33 19.047 50.725 34 19.806 51.966 35 20.569 53.203 PROCEDURE Prepare solutions quickly. From the time of introduction of samples into the neutron flux, the experiment takes approximately two and a half hours. You must arrive in the laboratory with a plan for how you will make your solutions (see below). Add all of your 10 mL unknown solution to a 100 mL volumetric flask. Rinse the vial with a few milliliters of deionized water and add the washings to the flask. Dilute to the mark with deionized water. Mix well. Using the 2.43E-02 g/mL manganese stock solution, prepare at least five (preferably six) calibration solutions. Start with zero concentration (blank) and do not exceed 5.00E-03 g/mL manganese in the most concentrated solution. Prepare a further 2.5-fold dilution of your unknown. In each volumetric flask, also add 5 mL of methanol, which acts as an antifreeze 92 agent. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 25mL volumetric flasks are provided. Ensure each solution is well mixed. Transfer 10.00 mL of solution from each flask to seven plastic scintillation vials. Before irradiating your samples, familiarize yourself with the proper procedures outlined in Appendix 1. Use the Am-Be neutron source to irradiate the samples for about 5000 seconds. During the irradiation period use the Perkin Elmer TriCarb 2910 scintillation counter to perform a Poisson test. Measure the activity of the tritium standard in a series of 25 time intervals each of 0.5 minutes (see appendix 2). Note that the default output for this instrument is in units of counts per minute. You must convert the data to the actual number of counts in 0.5 minutes. For the following statistical analysis the distribution of counts obtained in one minute intervals is not the same as that for 2 x (the counts obtained in 0.5 minute intervals). You can open the datafile output in spreadsheet software. Calculate the sample mean and the sample standard deviation s x . s x should be about equal to x 1/2 . Calculate the index of dispersion, D. For a Poisson distribution, D should be about N – 1 = 24. If the distribution is apparently Poissonian, continue with the next part of the experiment. If not, investigate the possible causes. Consider the impact of chosen confidence limits. Remove the vials from the neutron source after 5000 seconds. By automatic operation of the scintillation counter (see appendix 2) measure the Cerenkov radiation of the vials using counting periods of 10 minutes. The instrument will print out the elapsed times automatically. Once counting is complete, dispose of all samples into the holding tank provided. Note that the instrument scales the data to counts/min (CPM). Correct each sample count for background. Use the total elapsed time at which each sample was counted to correct for the exponential decay that occurred between the counting of the samples. The resulting counts are proportional not only to the concentration of manganese, but also to the neutron flux density. As flux density φ w varies slightly with the geometry of each activation well, it is necessary to normalize the counts to a common flux densityφ . The ratios of the well flux densities as compared to the average density are listed in the following table: WARNING: Radioisotopes, such as those created in this experiment, demand specific safety protocols. Their hazards and thus protocols vary with the nature of the radioactive decay. 56 Mn emits beta particles, a form of ionizing (thus tissue damaging) radiation. Beta particles are easily blocked, thus the greatest safety concern is to skin exposure. Wash off with soap and cold water in the event of skin exposure. Wear gloves (provided) whenever handling 56 Mn samples. Inform your TA and instructor immediately if spillage occurs. 93 Well No. φ φ w φ φ σ w 1 1.01 3 0.00 7 2 0.98 9 0.00 9 3 0.97 7 0.01 3 4 0.97 5 0.01 5 5 1.05 8 0.01 3 6 0.97 8 0.01 1 7 1.00 9 0.01 3 Normalize the counts for each sample, then determine the concentration in g/mL of manganese in your original unknown solution as received in the vial. IN YOUR REPORT What element is the decay product of 56 Mn? Include all the nuclear reaction equations for the processes necessary to this experiment. (Example: radium decay can be written as: 226 Ra → 222 Rn + 4 α) Describe how the scintillation counter works; what is it that is actually counted? Describe how the Am-Be source produces neutrons. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6 th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 32. 2. Ehman, W.D.; Vance, D.E. Radiochemistry and Nuclear Methods of Analysis. In Chemical Analysis, Vol. 116, Winefordner, J. D.; Kolthoff I. M., Eds.; Wiley: New York, 1991. 3. L’Annunziata, M.F. Ed. Handbook of Radioactivity Analysis; Academic Press: San Diego, 1998. (QC795 .H36 1998 in IKBLC) 94 APPENDIX I: Sample Irradiation with the Am-Be Source The samples are activated with an americium-beryllium neutron source of 1 curie activity. This is an "alpha neutron" source in which americium is the alpha emitter, and beryllium is the target material from which neutrons are emitted. Paraffin oil and wax are used to shield the laboratory from the source. Note: In the following procedure it is important to record the number of the well used for the activation of each sample. Attach a Plexiglas rod with a Velcro™ tip to the lid of each sample vial. Carefully align one sample vial in the top of each activation well, then simultaneously lower the vials into the neutron flux. Start a timer and activate the samples for at least 5000 seconds. Handle the activated samples with rubber gloves. After the samples have been activated, simultaneously lift the vials from the wells with the Plexiglas rods. Remove the rods and insert the vials into consecutive holes of the conveyor of the scintillation counter. Count the samples sequentially as described in Appendix 2. Once all samples have been counted, pour the activated solutions into the waste bottle provided. The isotopes will be allowed to decay for at least 10 half-lives before disposal of the solutions. "Wipe tests" are legally required on a daily basis to check laboratory work areas for contamination by radioactive species. The activation products of the 1-curie neutron source used in this laboratory do not produce detectable levels of high energy gamma radiation. However a simple test can still be made for contamination by activation products such as 56 Mn. Upon completion of your experiment wipe the working area with a "Kim Wipe" tissue dampened with methanol/water. Place the test tissue into a scintillation vial, and add water and methanol. Similarly prepare a blank sample vial containing a fresh tissue, methanol, and water. Measure the Cerenkov radiation of the vials using a 1 minute counting period. Counts of less than 100 per minute above background are considered to be indicative of low-level (acceptable) contamination. Report the test results on the posted table. APPENDIX II: Operating Instructions for the Perkin Elmer Tri-Carb 2910 Scintillation Counter Note: this instrument is quite new; it was purchased in October 2010 at a total cost of about $75,000. It is used for both teaching and research within the department. Like most other counters, this instrument is capable of handling a large number of samples simultaneously, but counting must be done sequentially. ‘Cassettes’ holding sample vials are moved around the deck of the counter. Each cassette is associated with an assay (a set of counting instructions) by the use of a ‘protocol flag’. The flag is a black plastic label that sits on the left side of the cassette, as shown in Fig. 2. The flag can be set so that the instrument counts the samples or so that the instrument bypasses counting that cassette. To set the flag for counting, gently push the tab to the left. 95 Figure 2. PE TriCarb Counter Cassette COUNTING TRITIUM DECAY Place the tritium ( 3 H) vial into the leftmost position in the cassette, then open the lid of the instrument and place the cassette on the right-hand side, parallel to the front of the instrument. Ensure the flag is set for counting. On the left-side of the main software screen, choose the flag number of your cassette, then right-click and choose Associate Assay. Select 3H_Chem_3XX.isa and click OK. This assay will count the sample for 0.5 minute intervals, 25 times. Data will be printed and also saved to a datafile. To start the counting process, click the green flag button . If you need to stop the counting prematurely, click the checkered flag button. AUTOMATED COUNTING OF MANGANESE SAMPLES Once the 3 H counting is finished, remove the tritium sample vial from the cassette and place it in the middle storage section of the instrument. Load your samples sequentially into the cassette, starting with the least concentrated sample in the leftmost position. Your unknown sample should be in the middle of the group and your blank sample in the rightmost position. Set the cassette flag for counting by sliding it gently to the left. An assay has been developed for you to use for counting manganese decay, but you should open it to check that the parameters haven’t been changed accidentally. Click File then Open Assay, followed by choosing 56Mn_Chem_3XX.isa. The assay type should be CPM. Click Count conditions and then verify the following settings: 96 Radionuclide: Mn56 Count Mode: High Sensitivity Pre-count Delay 0.00 min Count Time 10.00 min Quench Indicator SIS Assay Count Cycles 1 Repeat Sample Count 1 #Vials/Sample 1 2 Sigma % Terminator should be set to ‘Any region’ and 4.50%. This sets the counting time to be a maximum of ten minutes but it will stop counting early if the two standard deviation uncertainty of the measurement falls below 4.50%. Click Count Corrections and verify the following settings Static Controller √ Coincidence Time 18ns Luminescence Correction √ Delay before Burst 75ns Apply Half-life Correction should be OFF (box not checked) Click OK after verifying these parameters. If any parameters need to be changed, consult your TA or the lab director. On the left side of the main screen, choose the flag number of your cassette, then right-click and choose Associate Assay. Select 56Mn_Chem_3XX.isa and click OK. Once your sample vials have been placed in the cassette and the tab set, use the software to initiate counting: on the left side of the main screen, choose the flag number of your cassette, then click the green flag button in the top left corner to start the measurements. Data will be printed and also saved to a datafile. If you need to stop the counting prematurely, click the checkered flag button. 97 A-13 Determination of Nitrite and Nitrate by Ion Chromatography LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the processes that allow ion separation in ion chromatography Describe the components of the instrument including the pumping system, the column and the detector system Explain the advantages and disadvantages of using peak height compared to peak area as your analytical signal Recognize the general lab safety concerns and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of NO 2 - and NO 3 - in your unknown using the calibration curve method after applying appropriate dilution factors Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks, etc.) through practice BACKGROUND Ions in solution may be separated and quantified using ion exchange chromatography, an HPLC technique in which the stationary phase is a weak ion exchange resin. Resins made up of negatively-charged groups (for cation exchange) and of positively-charged groups (for anion exchange) have been developed. In this experiment the column packing is a low-capacity surface-sulfonated styrene-divinylbenzene resin to which have been bonded aminated latex beads. The eluant is a carbonate/bicarbonate buffer. The anions of the analyte and the bicarbonate buffer are in equilibrium with the aminated sites of the resin as follows: resin-N + HCO 3 - + Na + X - º resin-N + X - + Na + HCO 3 - where X - is an analyte anion. Separation of the analyte anions depends upon their relative affinities for the aminated sites. Ions are conveniently detected using a conductivity detector. To enhance the measurement it is necessary to suppress the high background conductivity of the carbonate/bicarbonate eluant. A cation exchange device between the separator column and the detector converts the bicarbonate anions of the buffer to carbonic acid, a weak electrolyte of low conductivity (<20µS), and the analyte anions to strong acids of high conductivity (i.e. sulfuric acid, nitric acid etc.). Whereas 98 conventional ion exchange columns become saturated and require periodic regeneration, the ion chromatograph is equipped with a membrane suppressor that is regenerated continuously. The eluant and sample anions flow in one direction over a cation-permeable membrane, while a regenerating solution of sulfuric acid flows continuously and simultaneously in the opposite direction over the opposite side of the membrane. The concentrations of nitrite and nitrate anions in solution are quantified simultaneously using this technique. Other analytical methods for determining nitrogen speciation typically involve the elemental analysis of total nitrogen followed by a separate analysis of one of the anions, and the determination of the second anion by difference. The advantages of the analysis by ion chromatography soon become apparent. PROCEDURE Add all of your unknown solution to a 100 mL volumetric flask. Rinse the vial with a few millilitres of deionized water and add the washings to the flask. Dilute to the mark with deionized water. Mix well. To seven 50 mL volumetric flasks add solutions containing 1.00E-04 g/mL nitrite, 2.00E-04 g/mL nitrate, and diluted unknown as follows: Flask mL NO 2 - (1.00 E-04 g/mL) mL NO 3 - (2.00 E-04 g/mL) mL diluted unknown Dilute to (mL) 1 0 0 0 50.00 2 1.00 1.00 0 50.00 3 2.00 2.00 0 50.00 4 3.00 3.00 0 50.00 5 4.00 4.00 0 50.00 6 5.00 5.00 0 50.00 7 0 0 10.00 50.00 Refer to Appendix I for operating instructions for the Metrohm Ion Chromatograph. WARNING: Sulfuric acid solution is used to regenerate the suppressor. Sulfuric acid is corrosive and acts as a strong oxidizer. Avoid skin contact. Rinse immediately with cold water if it occurs. 99 IN YOUR REPORT Compare the result for peak height vs. peak area; justify in the report your choice of peak area or height result. Report only one result for [nitrate] and one for [nitrite] on the first page. In comparing your results, make sure to use the appropriate statistical tests to determine whether the results agree. Discuss the principles of ion chromatography thoroughly. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6 th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 28 (especially 28F). 2. Harris, D.A. Quantitative Chemical Analysis, 8 th ed.; W.H. Freeman: New York, 2010; chapters 25-1 and 25-2. 3. Fritz, J.S. Ion Chromatography, Wiley-VCH: New York, 2000. (QD79.C453 G52 2000 in IKBLC) 4. Haddad, P.R. Ion Chromatography: Principles and Applications, Elsevier: New York, 1990. (QD79.C453 H33 1990 in IKBLC) 100 APPENDIX I: Operating Instructions for the Metrohm 883 Ion Chromatograph Overview: Transfer ~15 mL of the diluted unknown solution (i.e. flask 7 solution) into a 25 mL beaker. Follow the instructions below and start by running chromatograms of the following solutions: • the diluted unknown, • the diluted unknown solution to which has been added ~1 to 2 mL of a solution containing 1.00 E-04 g/mL nitrite, and • the diluted unknown to which has been added ~1 to 2 mL of a solution containing 2.00 E-04 g/mL nitrate. This technique is called “spiking” and is used to identify which chromatographic peak is due to the NO 2 - and which due to the NO 3 - . Run a chromatogram of each standard solution (flasks 1 to 6) and three duplicate chromatograms of the unknown solution (two additional runs if your first one looked ok). Integrate the peaks in each chromatogram and tabulate in your lab notebook data for each run. Data should include: retention time, peak height and peak area for each of nitrate and nitrite. Use both the peak height and peak area data to calculate the concentrations in g/mL of nitrite and nitrate in the original sample vial. (Note: Inspect the plots carefully before deciding upon the best method of calculation.) Instrument Use This instrument is fully-computer controlled. To begin, ensure the instrument itself is powered on by verifying a green light is present at bottom left of the instrument front panel. If not, the power switch is located on the rear. Load the ‘MagicIC’ software from the desktop icon. Start the instrument hardware by clicking the green ‘Start HW’ button as shown in the Figure 1 below. This starts the flow of both eluent and acid-regenerant solution. The conductivity should settle to a baseline of between 15-20 :S/cm with ‘dips-and-rebounds’ every 10 minutes of inactivity. Under the ‘Single Determination’ tab, select ‘CHEM3XX_A13’ as the method to use. This loads basic experiment parameters: eluent flow rate, column type, etc. In the ‘Remark’ field, enter your initials. Provide a sample name in the ‘Ident’ field. At the front of the instrument, remove the tubing from the volumetric flask of water, wipe it with a clean kim-wipe and then insert the tubing into your beaker of sample. 101 Figure 1. MagicIC Software Layout Click the ‘Start’ button. You now have 1 minute to fill the sample loop of tubing with the solution from your beaker. Do to this, pull gently on the syringe. The sample loop has a volume of 20:l, so drawing up 2 ml into the syringe is adequate to rinse and fill the tubing and the loop. Do not allow the end of the tubing to come out of your solution. Remove pressure from the syringe but leave the syringe in place. At the end of the 1 minute period, the instrument will automatically re-route eluent flow through the sample loop, pushing the sample onto the column. Each run will take approximately 15 minutes. To evaluate the data, select the ‘Database’ icon at left. Choose the datafile you wish to process; the most recently saved file is at the bottom of the list. Select ‘Determinations’ then ‘Reprocess’ from the top menu. The reprocessing window opens with four panels. The bottom right panel shows the chromatogram. To zoom into the data, drag a box around the area of interest. To zoom out, right-click and select ‘Show all’. The bottom left panel describes the parameters that will be used in the processing. Select ‘Integration’ and set ‘Smoothing’ to 20 and ‘Sensitivity’ to 30. These are reasonable starting values for integrating your chromatographic peaks; you may need to adjust these later. Click the ‘Components’ button and enter as many components (named as 1,2,3,…) as you have peaks. Enter the retention time for each peak. Click ‘Update’ to integrate your chromatogram. The table at top right displays peak data for each component: retention time, peak height, and peak area. Print two chromatograms: 1) An overlay of each of your standard solution runs. To do this, select all the relevant datafiles, then choose ‘Determinations’, ‘Overlay curves’ then select ‘Print PDF’. Once the pdf file is 102 created, you can print a hardcopy. If you are having trouble selecting datafiles, CONTROL + click on the time the determination started. 2) A chromatogram overlaying your three determinations of your unknown. Proceed as above, printing one copy of the pdf file that is generated. Shutting Down the System: After your determinations are complete: • Flush the sample introduction tubing by starting a run of deionized water. Fill the sample loop as before. After the instrument has injected the sample, you can stop the run. • Leave the instrument on for at least 10 minutes before shutting down the hardware (red ‘Stop HW’ button on the workspace screen). • After shutting down the hardware, power it off and shut-down the computer and monitor. 103 A-14 Determination of Biphenyl and p-Terphenyl by Liquid Chromatography LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the processes that allow analyte separation in reverse phase liquid chromatography Describe the components of the instrument including the pumping system, solvent choice, the column and the detector system Explain the advantages and disadvantages of using peak height compared to peak area as your analytical signal Recognize the safety concerns associated with methanol use and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of biphenyl and p-terphenyl in your unknown using the calibration curve method after applying appropriate dilution factors Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks, etc.) through practice BACKGROUND Involatile, non-polar compounds such as aromatic hydrocarbons can be determined by reverse- phase high performance liquid chromatography (RP-HPLC). In reversed phase chromatography the stationary phase is non-polar (the typical column material consists of silica gel with octadecylsilane (-Si-C 18 H 37 ) chemically bonded to the surface) and the mobile phase is relatively polar. The eluents in this experiment will consist of mixtures of methanol and water. The retention times for species eluting from the column depend upon the nature of the compounds, the column packing, and the eluent composition. Elution with a single solvent composition ("isocratic elution") may adequately separate certain species while leaving others unresolved. This is an example of the so-called ‘general elution problem’. In liquid chromatography, peak shape and separation can be improved significantly by changing or "ramping" the composition of the solvent during the course of the elution, a technique called “gradient elution”. Similar gradient-based techniques have been developed to address the general elution problem in other forms of chromatography. A Hewlett-Packard model 1100 liquid chromatograph will be used to analyze biphenyl and p- terphenyl in a complex solution matrix. Chromatograms prepared under isocratic conditions, one 104 with 90:10 (% volume) methanol:water and the second with 75:25 methanol:water, will be compared. Chromatographic conditions will then be optimized using gradient elution. Peaks of the analytes in the unknown mixture are identified by "spiking" the mixture with aliquots of the pure analytes in turn, and repeating the runs. Initially detection is by UV absorption at 254 nm. Once the peaks have been identified, the detector is programmed to monitor each analyte at its absorbance maximum (278 nm for p-terphenyl and 248 nm for biphenyl). PROCEDURE NOTE: Dispose of all methanol waste into the appropriately-labeled red plastic container in the fume hood. Using a dry 10 mL pipette cleaned with methanol pipette 10.00 mL from your unknown vial to a 50 mL volumetric flask. Dilute to the mark with methanol. To save time, proceed immediately to the following preliminary tests. The standard solutions described on the following pages can be prepared while the chromatograms are being run. NOTE: You MUST pipette 10.00 mL of the unknown solution – do not use all of it! Refer to Appendix I for operating instructions for the pumps, detectors, and data acquisition systems. Make the appropriate instrument settings (Appendix I(a)) and run a chromatogram of the unknown solution under isocratic conditions using 90:10 methanol:water as the eluent and a detection wavelength of 254 nm. Allow at least 5 minutes for all components to elute. Run a second chromatogram using 75:25 methanol:water. Allow at least 15 minutes for all components to elute. Refer to these chromatograms and program the pump to deliver an elution gradient as follows (Appendix I(a)(ii)): deliver 75:25 methanol:water for sufficient time to allow the first group of two or three major peaks to elute. (The first minor peak(s) are from the solvent.) increase ("ramp") the methanol content of the eluent during the next 2 minutes to a final composition of 90:10 methanol:water. deliver 90:10 methanol:water for sufficient time to allow the last peak(s) to elute. WARNING: Methanol is toxic primarily by ingestion and highly flammable. Keep bottles capped when not in use. Avoid skin contact and rinse with cold water if it occurs. Waste methanol must be placed in red solvent waste cans. Biphenyl and p-terphenyl are both skin/eye irritants and toxic by ingestion. Avoid skin contact with both. Wash with soap and cold water if it occurs. Both are harmful to aquatic life. 105 Run a gradient chromatogram of the unknown test sample under these conditions. Spike about 10 mL of the diluted unknown solution with ~1 mL of 2.00 E-05 g/mL biphenyl solution. Run a gradient chromatogram. Spike the same solution with 1 mL of 1.00 E-05 g/mL p-terphenyl solution. Run a gradient chromatogram. Compare the chromatograms and identify the peaks corresponding to the analytes. Finally, program the detector (Appendix I(b)(ii) or II(b)(ii)) to monitor each analyte at its optimum wavelength. Using the 2.00E-05 g/mL biphenyl and 1.00E-05 g/mL p-terphenyl stock solutions, prepare at least five (preferably six) calibration solutions. Start with zero concentration (blank) and do not exceed 2.30E-06 g/mL biphenyl and 1.25E-06 g/mL p-terphenyl in the most concentrated solution. All solutions should be prepared in HPLC-grade methanol. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 50mL volumetric flasks are provided. Ensure each solution is well mixed. Use the programmed instrument settings to run gradient chromatograms of the diluted unknown and solutions 1 to 6. Plot calibration curves of peak height and peak area vs. concentration of each standard. Calculate the concentrations of biphenyl and p-terphenyl in the original vial. IN YOUR REPORT Compare the result for peak height vs. peak area, report only one value on the first page, justify in the report. In comparing your results, make sure to use the appropriate statistical tests to determine whether the results agree. Discuss the principles of reverse phase high pressure liquid chromatography thoroughly. BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6 th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 28. 2. Harris, D.A. Quantitative Chemical Analysis, 8 th ed.; W.H. Freeman: New York, 2010; chapters 24. 3. Ahuja, S. Trace and Ultratrace Analysis by HPLC, Wiley: New York, 1991. (QD79.C454 A38 1991 in IKBLC) 4. Hanai, T. HPLC: A Practical Guide, Royal Society of Chemistry: London, 1999. (QD79.C454 H356 1999 in IKBLC) 106 APPENDIX I: Operating Instructions for the HP1100 HPLC System (a) Isocratic Elution with Detection at a Single Wavelength: The HP1100 system consists of four modules – the variable wavelength detector (VWD), the pump, the vacuum degassing module, and the solvent module. The modules are stacked vertically with the VWD on the bottom. Check that reservoir A is at least half-filled with deionized water, and reservoir B is at least half-filled with HPLC-grade methanol. If solvents must be added, get assistance! The solvents must be specially-filtered. Turn on the power switches at the bottom left of the detector module and the pump. The vacuum degasser is left on permanently. Turn on the computer. Ignore the request for a password (press “Cancel”). Double click the “LC ChemStation” icon to initialize the software. Check that the “Method and Run Control” window is opened and the following graphical representation of the instrument is displayed. If not, click “View” then select “Method and Run Control” and turn on “System Diagram”. The injector is at the left, followed by the pump, the column, the detector, and the acquired signal. Note: the order of this representation is incorrect! (In what way?) The remaining symbols represent data processing that will not be done in this experiment. In the following procedures the pump will be controlled by clicking ( ) with the mouse. Click ( ) to control the detector. The buttons will be used to ignite the deuterium lamp of the detector and turn on/off the pump. 107 (i) Setting the pump: Click ( ), then “Set up Pump…” to display the following window: In the “Control” box: • set Flow to “1.000 mL/min”. • set StopTime to “no Limit”. At this setting the chromatographic runs will proceed until they are stopped manually. • set PostTime to “Off”. The “post time” allows the eluent composition to restabilize after a gradient run. It is not required for isocratic elutions. In the “Solvents” box set the percent of solvent B: (methanol) at 90% or 75%. The percent of solvent A (water) will be set automatically. The Timetable box will be used later for defining elution gradients. For isocratic runs there should be no entries in the Timetable box. Click OK. (ii) Setting the detector: Click ( ), then “Set up VWD” signal…” to display the following window: 108 In the “Signal” box check that the Wavelength is set to “254 nm”. In the “Time” box set the Stoptime to “as Pump, no Limit”, and the Posttime to “Off”. These settings of the detector will not conflict with the time settings of the pump and will be used for the duration of the experiment. The “Timetable” box will be used later to program the detector to monitor each analyte at its optimum wavelength. Click OK. (iii) Running the chromatogram: To ignite the lamp and start the pump, click the on button at the right of the “Method & Run Control” screen. Do not switch the lamp off until the end of the experiment! Wait a few minutes for the displayed baseline to stabilize. Click RunControl at the top of the “Method & Run Control” window, then Sample Info… and make the appropriate entries into the following table: 109 At the start of the experiment enter: Operator Name: • your name Subdirectory: • C3XX Prefix: • your initials Counter: • 000000 Before running each new sample enter Sample Name and any Comments. Click OK. Rotate the injection valve lever to the 2:00 o'clock position. Use the 50 µL syringe to flush the valve 2 or 3 times. With the syringe needle still inserted in the valve, rotate the lever to the stop (at the 4:00 o'clock position). Remove the syringe. When a run is in progress, the chromatogram will be plotted in the signal box at the bottom left of the Method & Run Control screen. Click in the signal box to open a larger view of the chromatogram. Click the buttons in the bottom left corner of this new display to rescale the peaks. Once all peaks have eluted, click RunControl then “Stop Run/Inject/Sequence” to stop the run. Print the displayed chromatogram. Close the display of the chromatogram. 110 (b) Gradient Elution with Optimized Detection: (i) Programming the pump and running the chromatogram: In the next steps the pump will be programmed to elute 75:25 methanol:water until the first group of two or three peaks has just eluted. The eluent composition will then be ramped to 90:10 methanol:water over a 2 minute interval. This composition will be maintained until the last peak has eluted. • Click ( ), then “Set up Pump…” to display the window shown in Appendix II(a)(i). • In the “Solvents” window set initial composition of solvent B: (methanol) to 75%. • Click Append in the Timetable window. This opens the first row of the program timetable for the pump. Enter 0 min in the Time column. Enter 75% in the %B column. This timetable entry will set the initial eluent composition for all runs to 75:25 methanol:water. • Click Append to open the second row of the timetable. Refer to the test isocratic chromatogram that was run using 75:25 methanol:water. Our goal is to set the instrument to begin the gradient just after the first group of peaks has eluted from the column. You might expect that the best time to input is the time the absorbance returns to the baseline after the first group of peaks, but this is not the case. All chromatography instruments have a ‘dead time’ – the time required to pump through a non-retained species. In particular here, there is a finite time required for compounds to pass from the end of the column to the detector. You can subtract ~30s from the time that your absorbance returns to the baseline and use this as an estimate of the time those compounds eluted from the column. • Enter the time determined above. The gradient will be programmed to start at this time. Re-enter the initial % B (probably 75% methanol). • Click Append. Enter the time at which the gradient will end, approximately 2 minutes after the elution time of the first group of peaks. Enter the desired % B at the end of the gradient (probably 90% methanol). Click OK. Set the detector to monitor 254 nm or, if you have determined the elution order of the analytes, set it to monitor each analyte at its λ max (Appendix I(b)(ii)). Rotate the injection valve lever to the 2:00 o'clock position. Use the 50 µL syringe to flush the valve 2 or 3 times. With the syringe needle still inserted in the valve, rotate the lever to the stop (at the 4:00 o'clock position). Remove the syringe. Once all peaks have eluted, click RunControl then “Stop Run/Inject/Sequence” to stop the run. Print the displayed chromatogram. After the first gradient run click ( ), then “Set up Pump…”. Re-program the StopTime to terminate subsequent runs automatically after all peaks have eluted. Set PostTime to 3 min to 111 allot 3 minutes at the end of each run for the eluent to restabilize at its initial composition in readiness for the next run. (ii) programming the detector: Once the peaks for the analytes have been identified by spiking, and their retention times are known, the detector can be programmed to switch wavelengths at timed intervals so that each analyte is monitored at its optimum wavelength. Click ( ), then “Set up VWD” signal…” to display the window shown in Appendix II(a)(ii). • Refer to the test chromatograms of the spiked unknown sample. In the “Signal” box set the default Wavelength to the λ max of the analyte of interest that will elute first. • In the “Timetable” box click Append to open the first row of the timetable. Enter 0 in the Time column. In the Wavelength column enter the λ max of the analyte that will elute first. Turn on (√ √√ √ ) the Balance option to zero the baseline. • Click Append to open the second row of the timetable. Enter a time between the elution times of the two analytes of interest. Take care to program the wavelength to switch at a time when the signal is at baseline and is not indicating a peak. In the Wavelength column enter the λ max of the second analyte of interest. • Click OK. This completes the program. (c) Manual Reprocessing of Data: The computer selects peak baselines according to preset criteria, and calculates heights and areas based upon these baselines. These criteria may not be optimal for all peaks. It may be better to set the baselines of some peaks manually. Check the baseline processing of each chromatogram. If any require re-drawing, select View from the menu bar, then Data Analysis. Click Files, then Load Signal. Your files are in the [C3XX] directory. Check the file number that is printed on the top of the selected chromatogram, and open the data file. Get help to enable the manual re-draw button , as it was disabled when the program was started (an unavoidable nuisance!) Click then hold down the left button of the mouse and draw the baseline. Record the recalculated values for peak height and area on your original chromatograms. 112 (d) Shutting Down the HP1100: • Use the syringe to flush the injector 3 or 4 times with HPLC methanol. • Click View then select “Method and Run Control” to return to the main screen. Once all samples have eluted from the column, press the Off button to shut off the pump and the deuterium lamp. • Turn off the main power switches on the detector and pump. Do not turn off the power to the vacuum degassing unit! 113 A-16 Determination of Naphthalene by GC-MS LEARNING OBJECTIVES Before commencing the experiment, you should be able to: Describe the processes that allow analyte separation in gas chromatography Describe the components of the instrument including the injector, carrier gas, the column, vacuum system and detector system Explain the advantages and disadvantages of using peak height compared to peak area as your analytical signal Describe the reasons an internal standard is required for this GCMS method and explain the principles of choosing an appropriate internal standard Recognize the safety concerns associated with methanol use and plan to use best practices. On completion of the experiment and lab report, you should be able to: Correctly calculate the concentration of naphthalene in your unknown using the calibration curve and internal standard methods after applying appropriate dilution factors Apply fundamental statistics to collected data and calculate the confidence interval of your result Improve your accuracy and precision of liquid handling (pipeting, filling of volumetric flasks, etc.) through practice BACKGROUND GC-MS is a combined analytical technique whereby components in complex mixtures are separated chromatographically, then are detected by mass spectrometry. The separated compounds elute directly into the mass spectrometer where they are ionized, with the ions of the molecules and molecular fragments being counted and stored as a function of mass-to-charge ratio (m/z). Entire mass spectra are acquired at a rate of about two per second. The MS data can be re-processed and output in the following ways: “reconstructed ion chromatogram” (RIC) – a plot of the summed totals of the counts for all ions in each mass spectrum vs. time. RIC’s are equivalent to chromatograms output by conventional detectors such as FID’s or TCD’s. “extracted ion chromatogram” (EIC) – plots of counts vs. time using data selected at m/z ratios unique to particular analyte ions. EIC plots exclude the data for most non- analytes, thereby improving significantly the signal-to-noise ratios. EIC plots can also 114 resolve by mass those components that are not otherwise chromatographically resolved. This allows the use of deuterated isotopomers as ideal internal standards. mass spectra – plots of counts vs. m/z ratios of the ions of the eluted compounds. Mass spectra provide qualitative information about the analytes. Molecular structures can be determined directly from the m/z count patterns of the ions of the molecules and molecular fragments, or by comparison of the mass spectra with data stored in a library. Note: Your “Principle of Method” should include a functional description of the gas chromatograph and its interfacing with an MS detector. However, an outline of the ion trap mass spectrometer itself is provided here: The Varian GCMS used in this experiment is equipped with a capillary chromatographic column internally coated with 5% phenylpolysiloxane and 95% dimethylpolysiloxane (the stationary phase). The carrier gas is helium. Analytes elute directly from the column into the cavity of an ion trap mass spectrometer. This cavity is defined by a ring electrode and two cap electrodes. A 70eV electron beam from a heated filament ionizes the molecules. The ions initially are trapped in the cavity by the field of a radio frequency (RF) “storage” voltage applied to the ring electrode. The RF voltage is then ramped such that the ions, in order of increasing m/z ratio, oscillate axially towards the cap electrodes. At critical RF voltages the ion trajectories exceed the trapping field, and the ions are ejected – sequentially in order of increasing m/z ratio – through holes in the bottom end cap where they are detected by an electron multiplier and are counted as a function of m/z. Ion traps can be programmed to isolate ions of a single selected mass (m/z). These ions in turn can be further fragmented by “collision induced dissociation” (CID) to produce a second set of fragment ions which can be counted and stored as a second mass spectrum. This procedure, known as MS-MS, can be repeated sequentially. The analysis of successive mass spectra provides additional structural information about the original molecule. 115 PROCEDURE Before the laboratory period you should record into your notebook the structures and molecular weights of naphthalene and its fully-deuterated isotopomer, naphthalene-d 8 , which will be used as an internal standard. Without this information you cannot process your data. Using 1.00E-05 g/mL naphthalene stock solution prepare at least five (preferably six) calibration standard solutions. Start with zero concentration (blank) and do not exceed 1.20E-06 g/mL naphthalene in the most concentrated solution. Dilute your unknown solution five-fold. Add sufficient 5.00E-06 g/mL naphthalene-d 8 stock solution to make each solution (standards and unknown) 5.00E-07 g/mL in the deuterated compound. Dilute each solution to the mark with methanol. Pipets of 1, 1.5, 2, 2.5, 3, 4, 5 and 10 mL volume and 50mL volumetric flasks are provided. Ensure each solution is well mixed. NOTE: You MUST pipette 10.00 mL of the unknown solution – do not use all of it! Refer to appendix 1. Inject 1 µL samples and run chromatograms of the blank solution, the standards, and the unknown. Once all chromatograms have been run, refer to “TREATMENT OF DATA” and locate the peak(s) for naphthalene and naphthalene-d 8 . Construct EIC plots for naphthalene and naphthalene-d 8 in the standard solutions and the unknown. Determine the areas of the EIC plot peaks. Using naphthalene-d 8 as the internal standard, calculate the concentration of naphthalene in the original unknown solution. Use the software library search function to check the mass spectra corresponding to the peaks in the RIC chromatogram of the unknown sample. IN YOUR REPORT Thoroughly describe the principles and operation of both the separation-side of the instrument (the GC) and the detection-side (the MS). Discuss the internal standard method, including why deuterated isotopomers are ideal internal standards for GC-MS. Identify the contaminants in your unknown and comment upon as many eluted species as possible. Suggest source(s) for the species corresponding to background peaks. Why does the naphthalene mass spectrum show a strong line for the molecular ion? Note that naphthalene is spelled with a ‘phth’. WARNING: Methanol is toxic primarily by ingestion and highly flammable. Keep bottles capped when not in use. Avoid skin contact and rinse with cold water if it occurs. Waste methanol must be placed in red solvent waste cans. Naphthalene is a skin/eye irritant and toxic (known carcinogen). Harmful to aquatic life. Avoid skin contact; wash with soap and cold water if exposure occurs. 116 BACKGROUND READING 1. Skoog, D.A.; Holler, F. J.; Crouch, S.R. Principles of Instrumental Analysis, 6 th ed.; Thomson Brooks/Cole: Belmont, CA, 2007; chapter 20 and 27. 2. Harris, D.A. Quantitative Chemical Analysis, 8 th ed.; W.H. Freeman: New York, 2010; chapters 21 and 23. 3. Grob, R.L. Ed. Modern Practice of Gas Chromatography, 4 th ed.; Wiley: New York. 2004. (QD79.C45 M63 2004 in IKBLC). 4. Watson, J.T. Introduction to Mass Spectrometry: instrumentation, applications and strategies for data interpretation, 4 th ed. Wiley: Hoboken, NJ. 2007. (QC454.M3 W38 2007 in IKBLC) 5. Hubschmann, H.-J. Handbook of GC/MS: fundamentals and applications. Wiley-VCH: New York. 2001. (QD272.C44 H83 2001 in IKBLC) 117 APPENDIX: Operation of the Varian Saturn GC-MS The instrument is turned on at all times. Initially the System Control window should be displayed with the DailyChecks method running as follows: NOTE: Care must be taken in using the GC-MS. MS detection is extremely sensitive, and a gas leak could damage the detector. Before using the instrument ask the TA or the lab director to do a routine check of the instrument. Use only the method file that has been set up for your experiment. This ensures the detector will be turned on only after air and solvent have eluted from the column. The method also sets a mass range that avoids detection of these peaks, and sets proper temperatures for the injector, oven, and detector. 118 The CHEM 3XX A16.mth file: sets the following automated sequence for separating and detecting naphthalene and the other compounds in the solution matrix: Initial settings – the GC oven is set at 55 °C. The injector operates in “splitless” mode with 1 mL/min of helium flowing through the injector into the column. 0 to 0.7 minutes – the automated sequence is initiated when the sample is injected. For the first 0.7 minutes the sample is vaporized in the 250 °C injector. The semi-volatile analytes concentrate at the head of the 55 °C column. 0.7 minutes – the injector switches to the “split” mode allowing ~80 mL/min of helium to flush residual solvent and analyte from the injector to a “split” vent. The 1 mL/min He flow through the column is maintained. This is known as an 80:1 “split ratio”. 0.7 to 4 minutes – the oven temperature starts ramping at a rate of 14 °C/min. The relatively low boiling solvent elutes first as the oven temperature increases. 4 minutes – the mass spectrometer is turned on only after air and solvent have fully eluted from the column. m/z ratios from 70 to 250 are monitored. 4 to 11.7 minutes – as the temperature increases, analytes elute sequentially and are detected. An RIC chromatogram is plotted. 11.7 minutes – the oven temperature reaches 210 °C and the run terminates. The mass spectrometer is switched off and the oven temperature starts cooling to 55 °C in preparation for the next run. You will use only two buttons on the main toolbar at the top of the display: Selects the System Control window shown above. This window is used to check the status of the instrument and to control the instrument during chromatographic runs in accordance with appropriate method files. Selects the MS Data Review window. Data can be re-processed after the runs have been completed. Mass spectra can be recalled, and analytes can be identified through library searches. Chromatograms can be constructed using the data for selected ions (EIC plots), and chromatographic peak areas can be determined. In the System Control window click “File”, “Activate Method”, select the CHEM 3XX A16.mth method file, and click “Open”. Click “Windows” then “3800.44-Equilibrating” to monitor temperature stabilization of the GC. Wait until “3800.44-Ready” is displayed. (If “2000.40-Not Ready” is still displayed, this indicates the mass spectrometer has not yet been turned on.) 119 Click “Inject”, “Inject Single Sample”. Enter the sample name in the “Sample Name” box in the “Inject Single Sample” dialog window. Click the “Inject” button and wait for the status indicator to change from “NOT READY” to “WAITING”. Thoroughly flush a 10 µL syringe several times with the sample solution. Remove the needle from the solution, fully depress the plunger, and then draw in 1 µL of air. Put the needle tip into the solution and withdraw the plunger to the 2 µL mark. Finally draw in a further 1 µL of air. The syringe barrel now contains 1 µL of sample solution separated from the plunger and the needle tip by two 1 µL plugs of air. IMPORTANT! Proper injection technique is critical, and it varies with the particular analyte. Solutions of naphthalene in a complex matrix should be injected at a slow consistent rate. Over an interval of ~2 seconds insert the needle into the front injector, then depress the plunger at a consistent rate over a time interval of ~2 seconds. The automated sequence will start. The chromatogram and/or the mass spectrum can be monitored in real time during the run. Choose “Chromatogram and Spectra” in the box at the left of the screen. Click “Hide Keypad” to expand the display. The run will be monitored as follows: While the run proceeds, use the cursor to expand the display of any peaks of interest in the chromatogram. (You can use the key to return the display of the full chromatogram.) Click then click with the cursor on the chromatographic peak. The corresponding mass spectrum 120 will be displayed. Locate the peak(s) for naphthalene and deuterated naphthalene keeping in mind their molecular weights. The run will terminate automatically once the data has been acquired and saved. Do not print the chromatogram at this time. Click “Show Keypad” or you will not be able to start another run. Similarly run chromatograms of the standards and the unknown solution. When all runs have been completed, click “File”, “Activate Method”, and activate the DailyChecks.mth file. Leave the instrument in this condition. TREATMENT OF DATA Click in the main tool bar to open the MS Data Review window. In the “Select Plot(s)” window open the data file for the RIC chromatogram of the unknown solution. Use the cursor to highlight and expand the first few peaks. Click with the cursor to select and display mass spectra at two or three points on each peak. Significant differences between mass spectra measured at different points on a chromatographic peak are indicative of co-eluting compounds with slightly different retention times. Similarly rescale and check the other peaks in the chromatogram. Locate the peak(s) for naphthalene and naphthalene-d 8 . The mass spectrum for each of these compounds should include a strong line at the m/z value corresponding to the molecular ion, where m/z = MW/(1 + ). (Not all mass spectra include strong lines for the molecular ions. Some compounds are highly fragmented during ionization, whereby the strongest spectral lines correspond to the m/z values of the fragment ions.) To construct a EIC plot for naphthalene, click in the Spectra window. Enter the m/z ratio of the strongest line in the mass spectrum (assuming the m/z value is unique to naphthalene). Click “Plot”. The EIC plot will be displayed below the original RIC chromatogram. Similarly construct a EIC plot for naphthalene-d 8 using the m/z value of the strongest line in its mass spectrum. The RIC chromatogram and the two EIC plots will be displayed as a vertical stack. Click “Quantitation”, “Integrate All Plots in Time Range”, then click “Print”, to print a peak area table. Close the report window. Click (“Print all chromatogram and spectrum plots”). In the “Plot Arrangement” box select the plots to be stacked vertically and printed in landscape format. Click “Print”, to print the plot. Similarly construct and print a set of EIC plots and a peak area table for each of the standard solutions. Tabulate all results. Using the data for naphthalene-d 8 as an internal standard, calculate the concentration of naphthalene in the original unknown solution. Re-open and compare the RIC chromatograms of the unknown and the blank. Determine which peaks represent background and which represent contaminant species in the unknown. Click on each peak to display the mass spectra of the eluted compounds. Click on the mass spectra to run library searches of the compounds. For each mass spectrum ten possible matches are displayed, as well as a statistical evaluation of the quality of each match. A correlation >900 is considered 121 to be excellent. Click in the “MS Data Review” toolbar to “Print the Active Chromatogram and Spectrum Plot”. In the “Plot Arrangement” box select the plots to be stacked vertically and printed in landscape format. Click “Print” to print the report. On the printout identify as many of the contaminant species as possible. Can you suggest the source(s) of some of these compounds? (Keep in mind that detection by mass spectrometry is VERY sensitive.) Furthermore, can you suggest the source(s) of the species corresponding to the peaks shown in the chromatogram of your blank?