A SEMINAR ON: ADVANCES IN NMRCORRELATION SPECTROSCOPY4/21/2012 Prepared By Mr Nandan V. Shinde .Damle M.pharm Q.A.(1st sem) Guided By Mrs.M.C 1 4/21/2012 NMR SPECTROSCOPY Based upon measurement of absorption of electromagnetic radiation in the radiofrequency region of roughly 4 to 600 MHz Nuclei of certain atoms are involved in the absorption process In order to cause nuclei to develop the energy states required for absorption to occur, it is necessary to place analyte in an intense external magnetic field. 2 THE BASIC IDEA OF nD NMR 4/21/2012 For a single uncoupled proton resonance P: Preparation E: Evolution M: Mixing D: Detection Amplitude modulation of a singlet resonance as a function of t1 (signal decays due to spin relaxation) FT t2 FT t1 3 a sample containing two uncoupled spins A and X. Contour Plot 4 . of offsets nA and nX. will produce 2D peaks at their corresponding chemical shift offsets in both dimensions (2D spectra are in fact 3D images).The concept can be expanded to 3 and n dimensions 4/21/2012 two-dimensional (2D) experiment threedimensional (3D) experiment In the previous slide two-dimensional experiment. during the evolution time t1 the magnetization will evolve into anti-phase components. Diagonal Peak Cross-Correlation Peak 5 .4/21/2012 If spins A and X are J coupled. each magnetization (A and X) will not only modulate with its own resonance frequency but they will also modulate with the resonance frequency of the coupled spin. A necessary condition for the magnetization magnetization transfer to takes place during the mixing pulse (M). giving origin to a cross-correlation peak between them. As a result. and multiple quantum spectroscopy (MQS) increase the information content. multiple quantum filtering (MQF). and spin topology filtration reduce the complexity. 6 . Relayed correlation. while exclusive correlation (E. Both avenues can lead to three-dimensional spectroscopy. total correlation spectroscopy (TOCSY).COSY).4/21/2012 Extensions of the standard COSY experiment. Hetero TOCSY . TOCSY.ADVANCE METHODS FOR STRUCTURE ELUCIDATION OF SMALL MOLECULES 2D NMR Spectroscopy We will focus mainly in 1H and 13C nuclei at natural abundance. HMQC. NOESY -HSQC) Correlations Through Space: -Homonuclear . Multiple quantum. NOESY-HSQC. Correlations Through Chemical Bonds: -Homonuclear ( COSY. although 2D NMR is not limited to these nuclei only. NOESY) -Heteronuclear (HSQC. The basic COSY experiment. Singlets suppressed. DQF-COSY Correlating coupled homonuclear spins. Additional information on magnitudes of coupling disavtgtants may be extracted from 2D peak fine structure. Higher-resolution display than basic COSY. Typically used for correlating protons coupled over two or three bonds but may be used for any high-abundance nuclide. 4/21/2012 8 . Typically used for correlating protons coupled over two or three bonds.Through Chemical Bond 2D Homonuclear Chemical Shift Correlations : COSY-90 Correlating coupled homonuclear spins. 4/21/2012 Contd…. Vicinal and geminal coupling relationships can be differentiated in some cases. Typically used for correlating protons coupled over two or three bonds but may be used for any high-abundance nuclide. COSY-β Correlating coupled homonuclear spins. hence also known as longrange COSY. 9 . Reduced 2D peak structure over basic COSY. Often used to identify proton correlations over many bonds (>3). Delayed COSY Correlating coupled homonuclear spins through small couplings. Typically used for correlating adjacent carbon centers at natural abundance but has extremely low sensitivity. Powerful technique for analyzing complex proton spectra.4/21/2012 Contd… TOCSY Correlating coupled homonuclear spins and those that reside within the same spin system but which may not share mutual couplings. INADEQUATE Correlating coupled homonuclear spins of low natural abundance. Employs the propagation of magnetization along a continuous chain of spins. 10 . 4/21/2012 Contd. ADEQUATE Correlating coupled homonuclear spins of low natural abundance.. but employing 1H excitation and detection for sensitivity improvement. 11 . primarily 13C– 13C. 4/21/2012 THE COSY SPECTRUM OF m-dinitrobenzene 12 . 4/21/2012 THE COSY SPECTRUM OF 1-Chloro 2-Propanol 13 . 4/21/2012 COSY Spectrum of 2.Nitro propane 14 . 4/21/2012 Applications: 1) To determine structure in organic and biological samples. 2) To determine each & every proton in beta sheet of the pancreatic trypsin. 15 . 4/21/2012 HETCOR Hetronuclear Correlation Spectroscopy The 2D NMR Spectra that displays 13C-1H shift correlations are called HETCOR. HETCOR shows coupling between protons and the carbons to which they are attached. 16 . 4/21/2012 THE HETCOR SPECTRA OF 1-Chloro 2-Propanol Chemical shifts δH δC 1.4-3.5 20 67 51 17 .9 3.2 3. 56 21 4.66 79 18 .4/21/2012 HETCOR Spectrum of 2 Nitropropane No δ1H δ13 C 1 2 1. 04 19 .09 1.52 4.92 1.4/21/2012 HETCOR SPECTRUM FOR ISO-PENTYL ACETATE NO.1 2. 1 2 δ13 C 23 25 δH 0.69 3 4 6 37 63 20. 4/21/2012 INADEQUATE Incredible Natural Abundance Double Quantum Transfer Experiment Correlating coupled homonuclear spins of low natural abundance. 20 . Typically used for correlating adjacent carbon centers at natural abundance but has extremely low sensitivity. 4/21/2012 21 . 4/21/2012 22 . 4/21/2012 23 . Correlates all protons within the same spin system . so it does not suffer from cancellation of anti-phase peaks under conditions of low digital resolution or large linewidths. m. replaces the single mixing pulse of the basic COSY experiment. a priori . 24 .Number of transfer steps associated with each crosspeak not known. The TOCSY sequence.A second significant feature of TOCSY that contrasts with COSY is that it utilizes the net transfer of in-phase magnetization. In these instances. The spin-lock mixing time. this feature makes TOCSY the more sensitive of these two methods.TOtal COrrelation SpectroscopY – TOCSY (Former HOHAHA) 4/21/2012 . Sample: Chentobiose Solvent: CDCl3 Spectrometer: AVANCE 400 Probehead: Inverse Broadband with z-Gradients TOCSY 4/21/2012 B 1 A 1 A Anomeric H B Anomeric H B 1H NMR A 1H NMR 25 . 19F. Experimentally robust sequence. 31P (an „inverse technique‟). 1H. 1H. Employs detection of high-sensitivity nuclide. Provides improved resolution over HMQC.g. e. Employs detection of high-sensitivity nuclide. 26 . 31P (an „inverse technique‟).g. 19F. so it is better suited for crowded spectra but can be more sensitive to experimental imperfections. well suited to routine structural characterization. HSQC (Heteronuclear Single Quantum Correlation) Correlating coupled heteronuclear spins across a single bond and hence identifying directly connected nuclei.4/21/2012 Through Chemical Bond 2D Heteronuclear Chemical Shift Correlations : HMQC (Heteronuclear Multiple Quantum Correlation) Correlating coupled heteronuclear spins across a single bond and hence identifying directly connected nuclei. e. most often 1H–13C. typically 13C.nuclide. so has significantly lower sensitivity than inverse techniques. Benefits from high resolution in 13C dimension. otherwise superseded by above methods. 19F. 27 . Employs detection of the lower. HETCOR (Heteronuclear Correlation) Correlating coupled heteronuclear spins across a single bond. so may find use when this is critical. 1H. Employs detection of high-sensitivity nuclide.4/21/2012 HMBC (Heteronuclear Multiple Bonds Correlation) Correlating coupled spins across multiple bonds. e. 31P (an „inverse technique‟). Most valuable in correlating 1H–13C over two or three bonds. H–X–Y Triple-resonance methods for correlating protons and a heteratom (Y) whilst using a second heteroatom (X) to either relay the correlations or edit the correlation spectrum.g. Essentially HMQC tuned for the detection of small couplings. Powerful tool for linking together structural fragments. but may fail for mid-sized molecules (zero NOE). 28 . Observes steady-state or equilibrium NOEs generated from the saturation of a target. Suitable only for „small‟ molecules (Mr << 1000). Observes transient NOEs generated from the inversion of a target.4/21/2012 Through Space 2D Homonuclear Chemical Shift Correlations : NOE difference Establishing NOEs and hence spatial proximity between protons. Suitable for „small‟ (Mr << 1000) and large molecules (Mr > 2000) for which NOEs are positive and negative respectively. Estimates of internuclear separations can be obtained in favourable cases. for which NOEs are positive. NOESY(2D or 1D) Establishing NOEs and hence spatial proximity between protons. Suffers from low sensitivity but 1H detected variants can help. HOESY Establishing heteronuclear NOEs and hence spatial proximity between different nuclides. Can provide useful stereochemical information when homonuclear NOEs are insufficient or inappropriate. Observes transient NOEs in the rotating-frame. Suitable for any molecule but often essential for mid-sized molecules. but is prone to interference from other mechanisms so requires cautious interpretation. 1H–13C. 29 . for example. NOEs are positive for all molecular sizes. Estimates of internuclear separations can be obtained in favourable cases.4/21/2012 ROESY(2D or 1D) Establishing NOEs and hence spatial proximity between protons. 30 .4/21/2012 EXSY (2D) Qualitative mapping of exchange pathways in dynamic systems when exchange rates are slow on the NMR chemical shift timescale. Quantitative data on exchange kinetics can be obtained in favorable cases. meaning separate resonances are observed for each exchanging species. 551-594 II.6th Edition .http://en.http://www.chem. Stanley Crouch. Francis X. Published by : „Books Cole‟ Pg no:. James Holler . 540-550 31 .4/21/2012 REFERENCES: I.es/inteRMNet/doc/chem843-5. Webster . George Kriz. Published By :Cengage lerning Pg No:. Douglas Skoog.Kalsi . India Edidition.Pavia . Published By :Willey Publications Pg No:. http://www.ua. 2007. Robert M. 406-409 III.342-350.250-275 IV.ca/facilities/NMR/nmr/webcourse/cosy.h tm VII.org/wiki/Correlation_spectroscopy V. Silverstein. P.S.queensu.Gary Lampmann. “Spectroscopy of Organic Compounds” 6th edition Published By: New Age Publication Pg No:.intermnet.“Spectrometric Identification of Organic Compounds” . “Instrumental Analysis”. “Introduction To Spectroscopy” 3rd edition.PDF VI.Donald J.wikipedia. Kshirsagar Sir (H. Department) Mr. Mrs.A. Q. M.C..O.4/21/2012 My Sincere thanks to…. Kandekar Junior research fellow (Chemistry Department . Mohali) & ALL MY BATCHMATES/COLLIUGES 32 .NIPER. Abhijeet Ladake Student M. Somnath G.A. S.J .D. Mr. 3rd Sem. Damle Madam (Vice-Principal) Mr.pharm Q. 4/21/2012 THANK YOU!! 33 . 4/21/2012 Any Questions? 34 .