1. 核磁共振光譜與影像導論
Introduction to NMR Spectroscopy and Imaging Lecture 05 Basic Two-Dimensional Experiments (Spring Term, 2011) (Fall Term, 2011) (Spring Term, 2014) (Spring Term, 2015) (Spring Term, 2016) (Spring Term, 2017) Department of Chemistry National Sun Yat-sen University

2. Basic Two-Dimensional Experiments
Why multi-dimensional?
Resolution
Multi-quantum coherences
Coherence transfer
Types of multi-dimensional spectra:
To Correlate
To Resolve
To Exchange (Diffusion)

3. 1D is not enough for large molecules

4. Resolution improvement by using higher dimensions
,jasncjkhdsjkjk;dfaigvjtreitgikvcxmlfdlkvfdklvkfdkmfvkdfkvfdd.,fvdvfdmgfvdklfkfdffd
2D:
,j s k g v k d v f d
n j l o g k l
a b n d f j k y
d f m n w s q
s h a g v m d f d

5. Resolution improvement by using higher dimensions

6. Resolution improvement by using higher dimensions
Hidden, complicated correlations can be
revealed through cross peaks in a multi-
dimensional spectrum, with which the
chemical bonding, spatial closeness or
dynamical processes may be indentified.

7. Why multi-dimensional?
Resolution
Multi-quantum coherences
Coherence transfer
Types of multi-dimensional spectra:
To Correlate
To Resolve
To Exchange (Diffusion)
structure
dynamics

8. From 1D to 2D

9. From 1D to 2D t2 t1

10. If you look at the same data matrix in a different way….
You also get a series of FIDs.

11. In either direction, you see FIDs
You really don’t know which is “acquisition
dimension”, Do you?

12. A 2D Spectrum: A Single Peak
FT2 t1 f2
FT1

13. F2
A 2D Spectrum t2
FT2
FT1 F1 t1

14. 2D FT

16. Overview: Basic 2D Experiments
Homonuclear experiments (proteins < 8 ~kDa):
COrrelation SpectroscopY (COSY) J couplings
Double Quantum Filter COSY (DQFCOSY)J couplings
TOtal Correlation SpectroscopY (TOCSY) Side chains
Nuclear Overhauser Effect SpectroscopY (NOESY)Distances …
Heteronuclear experiments (proteins > ~8 kDa, connectivity):
HETeronuclear CORrelation spectroscopy (HETCOR)
Heteronuclear Single Quantum Coherence (HSQC)
Heteronuclear Multiple Quantum Coherence (HMQC)

17. Typical 2D Lineshapes

18. Resolution
The resolution in the second dimension is determined the same way
as in 1D NMR.
The resolution in the first dimension is determined by the longest evolution
time in the first dimension (maximum of t1).
The total evolution time in the indirect dimension is equivalent to the total
acquisition time in the detection dimension.

19. Window and Zero-Fillingt2 t1
FT2
window（pink）
Zero-filling 20484096（brown）
256 FIDs F2
4096 points
Increased to 4096 FIDS
F2 （4096 points）
F1（4096points）
FT 1
Window in t1
（dashed pink）

22. COSY pulse sequence (top) with a presaturation pulse to suppress
water signal. Also shown in the diagram are the coherence transfer
pathway (middle) and phase cycling scheme (bottom).

23. Through Bonds: COSY F2 (ωB, ωA) (ωA, ωA) (ωA, ωB) (ωB, ωB) F1
Evolve on spin A (B) during the incremented delay t1
Partially transfer magnetization from spin A (B) to spin B (A) during mixing time
Detect signal during t2

24. + -
First diagonal peak F1

25. + -
second diagonal peak

26. + -
second cross peak (SI)
first cross peak (IS)

27. +- -+ -+ +- + - - + F2 Dashed arrow: former life (t1)
+ -
coherence transfer
from S to I F2
Dashed arrow: former life (t1)
Solid arrow: this life (t2)
coherence transfer
from I to S F1

34. Strychnine

35. 2D Correlation Spectroscopy (COSY) Experiment –magnetization transfer through scalar (J) coupling. No signal from correlations that are more than three bonds apart since 4J ≈ 0.

37. Cross-Peak Intensity and J Coupling
The relationship between the cross-peak intensity and the magnitude of the J coupling is rather complicated：
1. For large J couplings (J>10 Hz), cross-peak intensity does not depend on J coupling, but is affected by relaxation rates. Weak intensity is usually from fast relaxation in the first dimension.
2. When J coupling is small and the maximum of t1 is not long enough, the cross peaks may be weak, even vanish. Increasing maximum t1 can improve (the total experimental time is increased).

40. A 2D dispersive peak

41. A 2D absorptive peak

43. + -

45. Absorptive cross peaks
Dispersive diagonal peaks
Dispersive cross peaks
Absorptive diagonal peaks

46. Absorptive cross peaks

47. Absorptive cross peaks

48. Dispersive diagonal peaks

49. F E D C B A

52. 1 2 5 3 4 6 7 8 9 10 1 3 2 9 6
7 4 8 5 10

54. Absolute Value Display

55. The major drawback of COSY: cross peaks
near the diagonal line may be obscured by
diagonal peaks.

57. DQF COSY

62. The Advantages of DQF COSY
Having the same phase for both the diagonal and cross peaks;
Because the magnetization is detected in anti-phase, the multiplet structure is
retained with opposite phase. In other words, the two lines of a doublet are 180° out
of phase with respect to each other. While this can lead to cancellation in crowded
regions of the spectrum, it also allows for the easy identification of multiplets
(based on their square, or box, shape), and for measuring the size of the scalar
coupling constant connecting the two spins.
Another advantage of the DQF-COSY experiment is not so immediately obvious. In order
for a transition to create multiple quantum levels, and to survive a multiple quantum filter,
you need to have at least two spins or three spins for a 2Q or 3Q transition, respectively.
Thus, singlets are drastically reduced in intensity in a DQF-COSY spectrum.

63. The Disadvantages of DQF COSY
There are also disadvantages to the DQF-COSY relative to COSY. First, the sensitivity of DQF is about a factor of two lower than regular COSY. Second, MQ relaxation in 1Hs is very slow, so the experiments require a relatively long d1 relaxation delay.
Thus, while a good COSY spectrum could be generated in about 2-4 hours of data accumulation, a good DQF-COSY requires about hours to allow complete relaxation during d1.

64. COSY Family COSY COSY-45,COSY-90 R-COSY (LR-COSY) pQF COSY PE-COSY
MQ COSY
TOCSY
zCOSY,zzCOSY, J.R.COSY
…..

65. 3JHNα = 5.9 cos2Φ - 1.3cos Φ
3Jαβ = 9.5 cos2χ cosχ1+ 1.8

66. J Coupling Measurement
J coupling and peak intensity
J coupling and linewidth
J coupling and overlap

67. With D2O as solvent, NH2 and NH are not observed owing to exchange between D and H.

68. 2D-NMR COSY spectrum of Rhodococcus sp. ZS402 overlaid with medium
2D-NMR COSY spectrum of Rhodococcus sp. ZS402 overlaid with medium. Signals in orange are from the sample and signals in grey are from the medium.