A SEMINAR ON: ADVANCES IN NMRCORRELATION SPECTROSCOPY

4/21/2012

Prepared By Mr Nandan V. Shinde .Damle M.pharm Q.A.(1st sem)

Guided By Mrs.M.C

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.

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

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, a sample containing two uncoupled spins A and X, 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).

Contour Plot

4/21/2012

If spins A and X are J coupled, during the evolution time t1 the magnetization will evolve into anti-phase components. A necessary condition for the magnetization magnetization transfer to takes place during the mixing pulse (M).

As a result, 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, giving origin to a cross-correlation peak between them. Diagonal Peak

Cross-Correlation Peak

4/21/2012

Extensions of the standard COSY experiment. Relayed correlation, total correlation spectroscopy (TOCSY), and multiple quantum spectroscopy (MQS) increase the information content, while exclusive correlation (E.COSY), multiple quantum filtering (MQF), and spin topology filtration reduce the complexity. Both avenues can lead to three-dimensional spectroscopy.
6

ADVANCE METHODS FOR STRUCTURE ELUCIDATION OF SMALL MOLECULES

2D NMR Spectroscopy We will focus mainly in 1H and 13C nuclei at natural abundance, although 2D NMR is not limited to these nuclei only. Correlations Through Chemical Bonds:

-Homonuclear ( COSY, TOCSY, Multiple quantum, NOESY) -Heteronuclear (HSQC, Hetero TOCSY , HMQC, NOESY-HSQC, NOESY -HSQC)
Correlations Through Space: -Homonuclear

Through Chemical Bond 2D Homonuclear Chemical Shift Correlations :

COSY-90 Correlating coupled homonuclear spins. Typically used for correlating protons coupled over two or three bonds but may be used for any high-abundance nuclide. The basic COSY experiment. DQF-COSY Correlating coupled homonuclear spins. Typically used for correlating protons coupled over two or three bonds. Higher-resolution display than basic COSY. Additional information on magnitudes of coupling disavtgtants may be extracted from 2D peak fine structure. Singlets suppressed.

4/21/2012

4/21/2012

Contd.

COSY- Correlating coupled homonuclear spins. Typically used for correlating protons coupled over two or three bonds but may be used for any high-abundance nuclide. Reduced 2D peak structure over basic COSY. Vicinal and geminal coupling relationships can be differentiated in some cases.

Delayed COSY Correlating coupled homonuclear spins through small couplings. Often used to identify proton correlations over many bonds (>3), hence also known as longrange COSY.

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. Employs the propagation of magnetization along a continuous chain of spins. Powerful technique for analyzing complex proton spectra. INADEQUATE Correlating coupled homonuclear spins of low natural abundance. Typically used for correlating adjacent carbon centers at natural abundance but has extremely low sensitivity.

10

4/21/2012

Contd..
ADEQUATE Correlating coupled homonuclear spins of low natural abundance, primarily 13C
13C,

but employing 1H excitation and detection for sensitivity improvement.

11

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 H C

1.2
3.9 3.4-3.5

20
67 51
17

4/21/2012

HETCOR Spectrum of 2 Nitropropane

No

1H

13 C

1 2

1.56 21 4.66 79

18

4/21/2012

HETCOR SPECTRUM FOR ISO-PENTYL ACETATE

NO.
1 2

13 C
23 25

H
0.92 1.69

3
4 6

37
63 20.09

1.52
4.1 2.04

19

4/21/2012

INADEQUATE Incredible Natural Abundance Double Quantum Transfer Experiment

Correlating coupled homonuclear spins of low natural abundance. Typically used for correlating adjacent carbon centers at natural abundance but has extremely low sensitivity.

20

4/21/2012

21

4/21/2012

22

4/21/2012

23

TOtal COrrelation SpectroscopY TOCSY (Former HOHAHA)

4/21/2012

- Correlates all protons within the same spin system - Number of transfer steps associated with each crosspeak not known, a priori - A second significant feature of TOCSY that contrasts with COSY is that it utilizes the net transfer of in-phase magnetization; so it does not suffer from cancellation of anti-phase peaks under conditions of low digital resolution or large linewidths. In these instances, this feature makes TOCSY the more sensitive of these two methods.

The TOCSY sequence. The spin-lock mixing time, m, replaces the single mixing pulse of the basic COSY experiment.
24

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

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, most often 1H13C. Employs detection of high-sensitivity nuclide, e.g. 1H, 19F, 31P (an inverse technique). Experimentally robust sequence, 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. Employs detection of high-sensitivity nuclide, e.g. 1H, 19F, 31P (an inverse technique). Provides improved resolution over HMQC, so it is better suited for crowded spectra but can be more sensitive to experimental imperfections.

26

4/21/2012

HMBC (Heteronuclear Multiple Bonds Correlation)

Correlating coupled spins across multiple bonds. Employs detection of high-sensitivity nuclide, e.g. 1H, 19F, 31P (an inverse technique). Essentially HMQC tuned for the detection of small couplings. Most valuable in correlating 1H13C over two or three bonds. Powerful tool for linking together structural fragments. HETCOR (Heteronuclear Correlation)
Correlating coupled heteronuclear spins across a single bond. Employs detection of the lower- nuclide, typically 13C, so has significantly lower sensitivity than inverse techniques. Benefits from high resolution in 13C dimension, so may find use when this is critical, otherwise superseded by above methods. HXY 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.

27

4/21/2012

Through Space 2D Homonuclear Chemical Shift Correlations :

NOE difference Establishing NOEs and hence spatial proximity between protons. Suitable only for small molecules (Mr << 1000), for which NOEs are positive. Observes steady-state or equilibrium NOEs generated from the saturation of a target.

NOESY(2D or 1D)
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, but may fail for mid-sized molecules (zero NOE). Observes transient NOEs generated from the inversion of a target. Estimates of internuclear separations can be obtained in favourable cases.

28

4/21/2012

ROESY(2D or 1D)

Establishing NOEs and hence spatial proximity between protons. Suitable for any molecule but often essential for mid-sized molecules; NOEs are positive for all molecular sizes. Observes transient NOEs in the rotating-frame, but is prone to interference from other mechanisms so requires cautious interpretation. Estimates of internuclear separations can be obtained in favourable cases.
HOESY Establishing heteronuclear NOEs and hence spatial proximity between different nuclides, for example, 1H13C. Can provide useful stereochemical information when homonuclear NOEs are insufficient or inappropriate. Suffers from low sensitivity but 1H detected variants can help.

29

4/21/2012

EXSY (2D) Qualitative mapping of exchange pathways in dynamic systems when exchange rates are slow on the NMR chemical shift timescale, meaning separate resonances are observed for each exchanging species. Quantitative data on exchange kinetics can be obtained in favorable cases.

30

4/21/2012

REFERENCES:
I. Douglas Skoog, James Holler , Stanley Crouch, Instrumental Analysis, India Edidition, 2007, Published By :Cengage lerning Pg No:.551-594 II. P.S.Kalsi , Spectroscopy of Organic Compounds 6th edition Published By: New Age Publication Pg No:.342-350, 406-409 III. Robert M. Silverstein, Francis X. Webster ,Spectrometric Identification of Organic Compounds ,6th Edition , Published By :Willey Publications Pg No:.250-275 IV.http://en.wikipedia.org/wiki/Correlation_spectroscopy V. http://www.intermnet.ua.es/inteRMNet/doc/chem843-5.PDF VI.http://www.chem.queensu.ca/facilities/NMR/nmr/webcourse/cosy.h tm VII.Donald J.Pavia ,Gary Lampmann, George Kriz, Introduction To Spectroscopy 3rd edition, Published by : Books Cole Pg no:. 540-550
31

4/21/2012

My Sincere thanks to..

Mrs. M.C. Damle Madam (Vice-Principal) Mr. S.J .Kshirsagar Sir (H.O.D. Q.A. Department) Mr. Abhijeet Ladake Student M.pharm Q.A. 3rd Sem. Mr. Somnath G. Kandekar Junior research fellow (Chemistry Department ,NIPER, Mohali) & ALL MY BATCHMATES/COLLIUGES

32

4/21/2012

THANK YOU!!

33

4/21/2012

Any Questions?

34