2D NMR.pptx

Two dimensional 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.

1. 2D NMR PRESENTED BY, ARDRA KRISHNA P V M.Pharm 1 ST 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 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 2

3. HISTORY 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 3

4. PROBLEMS WITH 1D NMR 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 1D WITH 2D NMR A conventional 1 H 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 Pulse Sequence 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 7 RELAXATION DELAY PULSE COLLECT DATA (t 1 )

8. 2D NMR Pulse Sequence 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 8 PULSE PULSE RELAXATION DELAY WAIT (t 1 ) COLLECT DATA (t 2 )

9. 2D NMR Two dimensional 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. Horizontal and vertical axis calibrated as Chemical shift Two frequency axes represent chemical shift and one intensity Diagonal signals (peaks) – A B (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 NMR is 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. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 12

13. FOUR PERIODS IN 2D NMR 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 13 Preparation Evolution Mixing Detection d1 p1 p2 t 1 ( aq )t 2

14. The Preparation period , where a magnetization coherence is created through a set of RF pulses ; The Evolution period , a determined length of time during which no pulses are delivered and the nuclear spins are allowed to freely process (rotate); The Mixing period , Magnetization is transferred through bond (or through space or chemical exchange) 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 2D NMR 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 16

17. EXAMPLES OF 2D HOMONUCLEAR EXPERIMENTS COSY ( CO rrelation S pectroscop Y ) TOCSY ( TO tal C orrelation S pectroscop Y ) INADEQUATE ( I ncredible N atural A bundance D oubl E QUA ntum T ransfer E xpt ) NOESY ( N uclear O verhauser E ffect S pectroscop Y ) ROESY ( R otating frame O verhauser E ffect S pectroscop Y ) 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 17

18. EXAMPLES TO 2D HETERONUCLEAR EXPERIMENTS HSQC ( H eteronuclear S ingle Q uantum C orrelation) HMQC ( H eteronuclear M ultiple Q uantum C orrelation) HMBC ( H eteronuclear M ultiple B ond C orrelation) 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 18

19. HOMONUCLEAR 2D EXPERIMENTS : There are 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  4 J 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 H N  and H alpha  protons are of special importance because the  phi  torsion angle of the protein backbone can be derived from the  3 J coupling constant between them. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 19

20. 2D TOCSY : In the 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 : The NOESY 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/r 6 , 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,  15 N and  13 C 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  15 N and  13 C 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 2D NMR 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. 12-07-2022 DEPT. OF PHARMACEUTICS, KMCH COLLEGE OF PHARMACY 25