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2D NMR.pptx
- 1. 2D NMR PRESENTED BY, ARDRAKRISHNA P V M.Pharm 1ST year DEPT. OF Pharmaceutics KMCH College of Pharmacy KMCH COLLEGE OF PHARMACY MODERN PHARMACEUTICAL AND ANALYTICAL TECHNIQUES 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 1 PRESENTED TO, DR. A. RAJASEKARAN Principal & Professor Dept. of Pharmaceutical Analysis KMCH College of pharmacy
- 2. CONTENT HISTORY PROBLEMS WITH 1DNMR COMPARISON OF 1D AND 2D NMR INTRODUCTION PRINCIPLE TYPES APPLICATION REFERENCE 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 2
- 3. HISTORY 1938 Isidor Rabi- Nuclear Magnetic Resonance 1971 Jean Jenner - Two dimensional NMR - COSY 1976 Richard Ernst - First two dimensional NMR experiment 1980s - application of NMR to protein structures In 1991- Ernst won a Nobel Prize in Chemistry for his contributions to Fourier Transform NMR 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 3
- 4. PROBLEMS WITH 1DNMR OVERLAPPING LINKAGE BETWEEN HETERO- NUCLEI MULTIPLE QUANTUM COHERENCES CORRELATE TWO DIFFERENT CHEMICAL SHIFT 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 4
- 5. Why 2D NMR? • Improve dissolution and dispersion • Determine structure (especially for molecules too complicated for 1D NMR ) • Involves series of 1D NMR experiments • Connectivity between nuclei ( homo and/or hetero) through chemical bond 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 5
- 6. COMPARISON OF 1DWITH 2D NMR • A conventional 1H NMR has a frequency axis and intensity axis, 2D NMR spectra have two frequency axis and intensity axis. • A normal NMR spectrum is obtained by plotting amplitude against one frequency dimension(F1) • In 2D NMR the spectrum is obtained by plotting amplitude against two frequency dimension(F1 and F2). Every peak in 2D NMR spectrum has two frequency co-ordinates which corresponds to F1 & F2. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 6
- 7. 1D NMR PulseSequence 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 7 RELAXATION DELAY PULSE COLLECT DATA (t1)
- 8. 2D NMR PulseSequence 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 8 PULSE PULSE RELAXATION DELAY WAIT (t1) COLLECT DATA (t2)
- 9. 2D NMR • Twodimensional Nuclear Magnetic Resonance (2D NMR) refers to a set of multi pulse techniques which were introduced to overcome the complex spectra obtained with NMR. • It is a set of NMR methods which give data plotted in a space defined by two frequency axes rather than one. • Conventional NMR spectra (CW NMR or 1D- spectra) are plots of intensity vs. frequency; In two-dimensional spectroscopy intensity is plotted as a function of two frequencies, usually called F1 and F2. • In general, 2d’s can be divided into 2 types, • Homo-nuclear • Hetero-nuclear • Each type can provide either through – bond(COSY-type) or through space (NOESY- type) coupling information. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 9
- 10. 1. Horizontal andvertical axis calibrated as Chemical shift 2. Two frequency axes represent chemical shift and one intensity • Diagonal signals (peaks)–AB (coupling between nuclei separated by 2-3 bonds) • Yellow color X is called as cross signals/peaks same peak on each coordinate axis 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 10
- 11. PRINCIPLE • 2D NMRis Nuclear magnetic resonance spectroscopy in which we provide a 90° pulse to our sample which is placed in uniform magnetic field then we wait for (0ms-5ms) depending upon the experiment type then again we provide 90 ° pulse and this time we measure signal as FID. • As a result we get Two FID signals which are plotted with respect to time domain. • To get a meaningful data , we apply Fourier transform program on FIDs to get frequency in terms of ppm. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 11
- 12. 12-07-2022 DEPT. OFPHARMACEUTICS, KMCH COLLEGE OF PHARMACY 12
- 13. FOUR PERIODS IN2D NMR 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 13 Preparation Evolution Mixing Detection d1 p1 p2 t1 (aq)t2
- 14. 1. The Preparationperiod, where a magnetization coherence is created through a set of RF pulses; 2. The Evolution period, a determined length of time during which no pulses are delivered and the nuclear spins are allowed to freely process (rotate); 3. The Mixing period, Magnetization is transferred through bond (or through space or chemical exchange) 4. The Detection period, in which the free induction decay signal from the sample is observed as a function of time, in a manner identical to one-dimensional FT-NMR 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 14
- 15. FREE INDUCTION DECAY •The signals decay away due to interactions with the surroundings. • A free induction decay (FID) is the result. • Fourier transformation, FT, of this time domain signal produces a frequency domain signal. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 15 TIME FREQUENCY FT
- 16. TYPES OF 2DNMR Homonuclear Heteronuclear correlation 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 16
- 17. EXAMPLES OF 2DHOMONUCLEAR EXPERIMENTS a) COSY (COrrelation SpectroscopY ) b) TOCSY (TOtal Correlation SpectroscopY) c) INADEQUATE (Incredible Natural Abundance DoublE QUAntum Transfer Expt) d) NOESY (Nuclear Overhauser Effect SpectroscopY) e) ROESY ( Rotating frame Overhauser Effect SpectroscopY) 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 17
- 18. EXAMPLES TO 2DHETERONUCLEAR EXPERIMENTS a) HSQC (Heteronuclear Single Quantum Correlation) b) HMQC (Heteronuclear Multiple Quantum Correlation) c) HMBC (Heteronuclear Multiple Bond Correlation) 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 18
- 19. HOMONUCLEAR 2D EXPERIMENTS: Thereare three 2D spectra which are widely used for the structure determination of proteins with a mass of up to 10 kD: • 2D COSY • 2D TOCSY • 2D NOESY 2D COSY: • In the COSY experiment, magnetization is transferred by scalar coupling. Protons that are more than three chemical bonds apart give no cross signal because the 4J coupling constants are close to 0. Therefore, only signals of protons which are two or three bonds apart are visible in a COSY spectrum (red signals). The cross signals between HN and Halpha protons are of special importance because the phi torsion angle of the protein backbone can be derived from the 3J coupling constant between them. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 19
- 20. 2D TOCSY: • Inthe TOCSY experiment, magnetization is dispersed over a complete spin system of an amino acid by successive scalar coupling. The TOCSY experiment correlates all protons of a spin system. Therefore, not only the red signals are visible (which also appear in a COSY spectrum) but also additional signals (green) which originate from the interaction of all protons of a spin system that are not directly connected via three chemical bonds. • Thus a characteristic pattern of signals results for each amino acid from which the amino acid can be identified. However, some amino acids have identical spin systems and therefore identical signal patterns. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 20
- 21. 2D NOESY: • TheNOESY experiment is crucial for the determination of protein structure. It uses the dipolar interaction of spins (The nuclear Overhauser effect, NOE) for correlation of protons. The intensity of the NOE is in first approximation propotional to 1/r6, with r being the distance between the protons. The correlation between two protons depends on the distance between them, but normally a signal is only observed if their distance is smaller than 5 Å. • The NOESY experiment correlates all protons which are close enough. It also correlates protons which are distant in the amino acid sequence but close in space due to tertiary structure. This is the most important information for the determination of protein structures. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 21
- 22. HETERONUCLEAR NMR SPECTROSCOPY: •Apart from protons, a protein contains other magnetic active nuclei. For NMR of proteins, 15N and 13C are of special importance. The use of these hetero nuclei allows some new features in NMR which facilitate the structure determination especially of larger proteins. • The natural abundance of 15N and 13C is very low and their gyromagnetic ratio is markedly lower than that of protons . • Therefore, two strategies are used for increasing the low sensitivity of these nuclei: Isotopic enrichment of these nuclei in proteins and enhancement of the signal to noise ratio by the use of inverse NMR experiments in which the magnetization is transfered from protons to the hetero nucleus. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 22
- 23. APPLICATIONS OF 2DNMR • Structural identification in organic and biological chemistry. Since its creation, 2D NMR has been useful for elucidating the structure of small molecules. • Advanced computing power now allows the structure of large, biological molecules to be solved. • Used COSY and NOESY to obtain individual assignments for each proton in the protein backbone in the β-sheet secondary structure of pancreatic trypsin inhibitor. • COSY NMR was used to determine the J connectivities on the protein backbone. • 2D NMR uses a sequence of two pulses with a series of different evolution times to determine which nuclear spins are coupled to one another. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 23
- 24. REFERENCES • Ashutosh kar,Pharmacetical Drug Analysis, Revised Second Edition • D.A Skoog, F.J Holler and S.R Crouch, Principles of Instrumental Analysis, Sixth Edition • William Kemp, Organic Spectroscopy, Third Edition 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 24
- 25. THANK YOU ! 12-07-2022 DEPT.OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 25