1. Multipulse techniques
Introduction to 2D NMR
Multipulse techniques
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2. Organic Structure Analysis, Crews, Rodriguez and Jaspars

3. ONE-PULSE SEQUENCE
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4. ONE-PULSE SEQUENCE (90o)x 1H Preparation Detection
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5. BASIC LAYOUT OF A 2D NMR EXPERIMENT
Preparation
Evolution t1
Mixing t
Detection t2
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6. INVERSION-RECOVERY PULSE SEQUENCE
(180o)x
(90o)x t2 t1 1H
Preparation
Evolution
Detection
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7. INVERSION-RECOVERY PULSE SEQUENCEt1 t1
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8. SPIN-ECHO PULSE SEQUENCE
(90o)x
(180o)x t1 t1 t2
13C
Prep.
Evolution
Detection
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9. SPIN-ECHO PULSE SEQUENCE
FT gives
null signal
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10. 1H-1H COSY (COrrelated SpectroscopY)
(90o)x
(90o)x t2 t1 1H
Preparation
Evolution
Detection
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11. n is the number of increments
PROCESSING 2D DATA
n is the number of increments
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12. TYPES OF 2D NMR EXPERIMENTS
AUTOCORRELATED
Homonuclear J resolved
1H-1H COSY
TOCSY
NOESY
ROESY
INADEQUATE
CROSS-CORRELATED
Heteronuclear J resolved
1H-13C COSY
HMQC
HSQC
HMBC
HSQC-TOCSY
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13. AUTOCORRELATED EXPERIMENTS – 1H-1H COSY
f1=f2=diagonal
Gives:
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14. AUTOCORRELATED EXPERIMENTS – 1H-1H COSY
Organic Structure Analysis, Crews, Rodriguez and Jaspars

15. REQUIREMENTS FOR 1H-1H COSY
Number of transients required is half that needed to give decent 1D 1H NMR spectrum
Most of the time we use a ‘double quantum filtered COSY’ (DQF-COSY):
Same information as COSY but removes single quantum transitions (large singlet peaks from Me groups), meaning we can see things closer to the diagonal. Solves problems in case where there is a dynamic range problem (very large and very small peaks in same spectrum)
It is phase sensitive, we acquire 2 x number of increments (real and imaginary). Get coupling information from phases of correlation peaks.
Organic Structure Analysis, Crews, Rodriguez and Jaspars

16. PEAK PICKING FOR 1H-1H COSY
DQF-COSY
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17. PEAK PICKING FOR DQF-COSY
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18. TOtal Correlation SpectroscopY (TOCSY)
HOmonuclear HArtman-HAhn spectroscopy (HOHAHA)
Increasing the mixing time (30 – 180 ms):
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19. TOtal Correlation SpectroscopY (TOCSY)
HOmonuclear HArtman-HAhn spectroscopy (HOHAHA) dH dH
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20. TOtal Correlation SpectroscopY (TOCSY) HOmonuclear HArtman-HAhn spectroscopy (HOHAHA)
Like COSY in appearance
Relies on relayed coherence during spin-lock mixing time
The longer tmix, the longer the correlations (30 – 180 ms gives bonds)
Relays can occur only across protonated carbons – not across quaternary carbons (spin systems)
Very useful for systems containing discrete units eg proteins and polysaccharides
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21. NOESY (Nuclear Overhauser Effect SpectroscopY)
ROESY (Rotating Overhauser Effect SpectroscopY)
Through-space correlations
Up to 5 Å
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22. NOESY (Nuclear Overhauser Effect SpectroscopY)dH
MW = 300 Da
tmix = 800 ms dH
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23. ROESY (Rotating Overhauser Effect SpectroscopY)dH
MW = 800 Da
tmix = 300 ms dH
Organic Structure Analysis, Crews, Rodriguez and Jaspars

24. NOESY (Nuclear Overhauser Effect SpectroscopY) ROESY (Rotating Overhauser Effect SpectroscopY)
Give through-space correlations up to 5 Å
The effect relies on molecular size. The NOE effect ~ 0 at 1000 Da. It works well for small molecules (tmix ~ 800 ms) and macromolecules (tmix ~ 100 ms).
In the intermediate range use ROESY with tmix ~ ms
Both NOESY and ROESY need long relaxation delays (2 s)
True NOE and ROE peaks are negative. In NOESY can get COSY peaks showing (positive). In ROESY can get TOCSY peaks showing (antiphase).
To determine mixing time do inversion-recovery experiment to find average T1. As a rule of thumb, NOESY tmix = T1/0.7, ROESY tmix = T1/1.4
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25. INADEQUATE – Incredible Natural Abundance DoublE
QUAntum Transfer Experiment
13C-13C
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26. INADEQUATE – Incredible Natural Abundance DoublE
QUAntum Transfer Experiment dC dC
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27. INADEQUATE – Incredible Natural Abundance DoublE QUAntum Transfer Experiment
C-C correlation experiment
Relies on two 13C being adjacent.
Chance of 13C-13C = 1/10 000
Works by suppressing 13C single quantum signal (hence DQ)
Needs signal/noise of 25/1 with 1 transient 13C NMR experiment to get spectrum in 24 h
For compound of 150 Da, need 700 mg in 0.7 mL CDCl3 (~ 6M)
With low volume probes and image recognition software can get away with much smaller samples and poorer signal/noise
Organic Structure Analysis, Crews, Rodriguez and Jaspars

28. HETERO CORRELATED EXPERIMENTS (13C-1H) 13C DETECTED
1H-13C COSY (also called HETCOR). Two types:
Direct correlations (1JCH = 140 Hz) C-H
Indirect (long-range) correlations (2-3JCH = 9 Hz) C-C-H and C-C-C-H
Very insensitive
For J = 140 Hz take 1/3 number of transients needed to get 13C NMR spectrum with S/N = 20/1. If 300 transients for 13C NMR, 2D with 256 increments takes 14 h.
For J = 9 Hz take 1/2 number of transients needed to get 13C NMR spectrum with S/N = 20/1. Needs longer relaxation time (2s). If 300 transients for 13C NMR, 2D with 256 increments takes 32 h.
Outdated
Organic Structure Analysis, Crews, Rodriguez and Jaspars

29. HETERO CORRELATED EXPERIMENTS (13C-1H) 1H (INVERSE) DETECTED
Direct correlations (C-H, 1JCH = 140 Hz) obtained from HMQC or HSQC experiment (Heteronuclear Multiple/Single Quantum Coherence)
Indirect (long-range) correlations (C-C-H, C-C-C-H, 2-3JCH = 9Hz) obtained from HMBC experiment (Heteronuclear Multiple Bond Correlation). Set JCH to other values for certain systems.
These experiments are 1H detected and have inherent sensitivity advantage (gH = 4gC) Chance of 13C-1H is 1/100
With pulsed field gradients (PFG), it is possible to run 2D heterocorrelated experiments with single transients and 256 increments in 8-15 minutes!
Without PFG need to phase cycle to remove artefacts. (4 transients minimum: t = 30 min; but 64 for full phase cycle: t = 9h).
Organic Structure Analysis, Crews, Rodriguez and Jaspars

30. HSQC versus HMQC HMQC HSQC Absolute value
Half the resolution of an HSQC
Can alter pulse sequence to get HMBC
HSQC
Phase sensitive
Double the resolution of an HMQC
Can edit to get positive peaks for CH, CH3 and negative peaks for CH2.
Organic Structure Analysis, Crews, Rodriguez and Jaspars

31. HSQC – Heteronuclear Single Quantum Coherence
1JCH = 140 Hz; C-H direct correlations (1 bond)
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32. HSQC – Heteronuclear Single Quantum Coherence
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33. Edited HSQC – Heteronuclear Single Quantum Coherence
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34. HMBC – Heteronuclear Multiple Bond Correlation
2-3JCH = 9 Hz; C-H indirect (long range) correlations
(2-3 bonds) C-C-H & C-C-C-H
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35. HMBC – Heteronuclear Multiple Bond Correlation
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36. 3D Experiments – HSQC-TOCSY
Mixing time ms
3-7 bonds
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37. 3D Experiments – HSQC-TOCSY
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38. 3D Experiments – HSQC-TOCSY
3D experiment condensed into 2D.
Concatenation of HSQC and TOCSY pulse sequences
Sorts TOCSY correlations in spin system according to carbon chemical shift – increases resolution of TOCSY by adding 13C dimension
See direct (C-H) correlations as in HSQC, and long range correlations within spin systems depending on mixing time (30 – 180 ms, 3 – 7 bonds). Can’t go across quaternary C or heteroatom as it the TOCSY effect needs protons.
Very effective for modular systems with separate spin systems such as polysaccharides and peptides.
Organic Structure Analysis, Crews, Rodriguez and Jaspars

39. General procedure for running 2D spectra
Insert sample, tune 1H and 13C channels
Lock and shim (determine 90o pulse width)
Acquire 1H NMR spectrum
Change spectral window to ± 1 ppm of spectrum
Re-acquire 1H spectrum
Phase spectrum, apply baseline correction
Acquire 13C spectrum in optimum spectral window
Call up macro for 2D experiment. Use 1H and 13C parameters for 2D experiments
Alter number of transients, number of increments to fit the time available
Repeat steps 8 & 9 for other 2D experiments required
Set experiments running
Organic Structure Analysis, Crews, Rodriguez and Jaspars

40. Processing 2D spectra – Phase sensitive experiments (DQF-COSY, TOCSY, NOESY, ROESY, HSQC, HSQC-TOCSY)
Fourier transform the first increment
Apodise t2 using shifted sine bell squared
Fourier transform t2  f2 using apodisation function in 2.
Apodise t1 using shifted sine bell squared
Fourier transform t1  f1 using apodisation function in 4.
Phase spectrum in both dimensions if necessary
Organic Structure Analysis, Crews, Rodriguez and Jaspars

41. Processing 2D spectra – Absolute value experiments (COSY, HMBC)
Fourier transform the first increment
Apodise t2 using sine bell
Fourier transform t2  f2 using apodisation function in 2.
Apodise t1 using sine bell
Fourier transform t1  f1 using apodisation function in 4.
No phasing necessary
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42. APODISATION - Phase sensitive experiments
APODISATION - Absolute value experiments
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