1. Chapter 7 (HSQC/HMQC/HMBC)

2. Proton-X Nucleus Correlation
Correlate proton with the carbon or other X-nucleus that the proton is bound to in 2D.
Indirect detection of carbon experiment
-the experiment directly detects proton (detect dimension or t2; the fid is proton) and indirectly detects carbon (indirect detect dimension or t1).
Why would you want to correlate Proton chemical shift with X nucleus (usually carbon or nitrogen) chemical shift?
1) Overlap of proton resonances
2) Assignment of carbon (or nitrogen ...) spectra based upon proton spectrum or assignment of proton spectrum based upon carbon (or nitrogen ..)
3) Indirect detection of carbon (or nitrogen) is more sensitive than direct observation or carbon (or nitrogen).

3. Proton-X Nucleus Correlation
What are the advantages of 1D direct detection of carbon versus 2D proton-carbon correlation?
1) Carbons, with no protons attached, do not show up in direct proton-carbon correlations.
2) Resolution of 1D carbon detect should be better than 2D indirect detect of carbon.
3) 2D takes a little longer if sample is very concentrated

4. Sensitivity of H-X Correlations

5. 2D Proton-Carbon Correlation

6. HSQC/HMQC HSQC = Heteronuclear Single Quantum Coherence
HMQC = Heteronuclear Multiple Quantum Coherence
What is the difference between HSQC and HMQC?
The two pulse sequences are very different, one depends upon the build up of single quantum coherence (as in INEPT), the other multiple quantum coherence (as in DEPT), but the results look nearly the same to the user.
How do the experiments work?
You start with magnetization on the proton and then transfer the magnetization to the carbon, record the chemical shift of the carbon, transfer back to proton, and detect the magnetization on proton.

7. HMQC 1H 90ºX  180ºX  Acquire X 90ºX  t1  90ºX Decouple
HMQC Pulse Sequence:
1H 90ºX  ºX  Acquire
X ºX  t1  90ºX Decouple
= 1/2JCH = ~1/(140*2) for aliphatic = ~1/(200*2) for aromatic

8. HSQC/HMQC
HSQC is not affected by proton proton couplings during t1 as only X chemical shift evolves.
HSQC is more complicated pulse sequence; it is more susceptible to signal loss due to incorrect pulse widths, RF inhomogeneity, and off-resonance effects.

9. 2 Major Versions on HSQC for Small Molecules
1) Multiplicity edited HSQC (gHSQC)
2) Sensitivity Enhanced HSQC (gHSQCSE)

10. HSQC Parameters

11. HETCOR HETCOR- HETeronuclear shift CORrelation
H/X correlation with detection of the X nucleus rather than 1H, which obviously will be less sensitive due to the difference in the  for 1H and any other nucleus
The advantages of HETCOR over HMQC are:
1) Reduction in the amount of time that magnetization is in the x-y plane when it can be lost due to T2 relaxation
2) Reduction of noise in t1, but modern versions of the HMQC are not as bad as the original HMQC for t1 noise, so this point is minor
3) Resolution in 13C is better, as 13C is now the dimension detected (remember that np >> ni).
Of course, this means that resolution in 1H is much worse.
Generally, resolution in 1H is preferred over resolution in 13C as there is greater resonance dispersion in 13C than 1H.

12. HETCOR

13. HMBC
(Heteronuclear shift correlations through Multiple Bond Connectivities)
Proton resonances are correlated by direct coupling through multiple bonds to carbon resonances while attempting to filter out the 1-bond proton-carbon correlations.
Thus, the HMBC depends upon the magnitude of the 2JCH, 3JCH and 4JCH. As in the COSY type experiments, the three-bond and four-bond scalar coupling constants are dependent upon torsion angle, in this case that is the proton-carbon torsion angle.

14. HMBC
The one-bond carbon-proton couplings are supposed to be filtered out, but filtering of resonances is never 100%, so normally there are some weak 1-bond correlations observed between proton and carbon.

15. HMBC Parameters

16. Other Methods for Multiple Bond Correlation of 1H and X Nuclei
I. Heteronuclear COSY
This is a 1H detect experiment so it is sensitive, with antiphase multiplet crosspeaks as in DQF-COSY.
1H ºX Acquire
X 90ºX —— t1—— 90ºX Decouple

17. Other Methods for Multiple Bond Correlation of 1H and X Nuclei
The advantages of the COSY-type experiment versus the HMQC-type (HMBC) are:
1) There is no HMQC-type delay that is correlated to the coupling constant.
The crosspeak intensity is mostly correlated to the magnitude of the JHX coupling constant as in a COSY, and the T2 relaxation.
2) The JHX coupling constant is easily measured.
Since the 3JHX coupling constant has torsion angle information according to the Karplus relationship, precise measurement of the 3JHX can be useful data.
3) There is a greater loss of magnetization in the HMQC type experiment due to T2 relaxation because the HMQC delay is longer than the delays during the COSY type experiment.
The primary advantages of HMBC over the heteronuclear COSY:
1) Higher sensitivity overall, as the Heteronuclear COSY is a 1H detect experiment but the initial pulse is on X.
2) Difficult to remove 1-bond HX couplings in heteronuclear COSY

18. II. CIGAR (Constant time Inverse-detected Gradient Accordion rescaled long-range heteronuclear multiple bond correlation)
Modified HMBC type experiment to achieve better resolution in t1 and improve the signal/noise for crosspeaks with coupling constants different than input value.
Advantages of CIGAR over HMBC:
1) Somewhat better resolution in t1 dimension
2) Improved signal/noise for crosspeaks with coupling constants different from input value
3) Can potentially observe difference in 2J vs. 3J
Disadvantages:
1) Overall signal/noise is poorer for CIGAR
2) T2 relaxation is a bigger problem as the CIGAR pulse sequence is longer

19. HSQC-TOCSY
First, an HSQC correlating the proton and nitrogen or carbon, then a 1H/1H TOCSY.
When there are resonances with coincidental chemical shifts in proton but not carbon, you can acquire a HSQC-TOCSY and determine which resonance is which.
This can also be acquired as 3D proton/carbon/proton as well.

20. HSQC-TOCSY
First, HSQC, record 13C shift
Then, TOCSY, detect proton

21. HSQC-TOCSY on Sugar

22. Inadequate