Decoupling in 2D HSQC Spectra

HMQC and HSQC NMR data are commonly used to correlate the chemical shifts of protons and ¹³C (or ¹⁵N) across one chemical bond via the J coupling interaction.  The data are ¹H detected, with the ¹H chemical shift in the horizontal F2 domain and the ¹³C (or ¹⁵N) chemical shift in the vertical F1 domain.  In the case of ¹H and ¹³C, the technique depends on protons bonded to ¹³C.  ¹H–¹²C spin pairs provide no coupling information and are suppressed by the method.  If one is to observe the ¹H signal of a ¹H-¹³C spin pair, one expects to observe a doublet with splitting ¹J_H-C (i.e. the ¹³C satellites).  Likewise, if one is to observe the ¹³C signal of a ¹H-¹³C spin pair, one expects to observe a doublet with the same splitting.  2D HSQC spectra are normally presented with both ¹H and ¹³C decoupling yielding a simplified ¹H-¹³C chemical shift correlation map over one chemical bond.  The figure below shows one of the most commonly used gradient HSQC pulse sequences.  The ¹H and ¹³C decoupling elements of the sequence are highlighted in yellow and pink, respectively.

During the evolution time, t1, the ¹³C chemical shift and ¹H-¹³C coupling evolve.  The ¹H 180° pulse (color coded in yellow) in the center of the evolution time refocuses the coupling and as a result decouples protons in the F1 (¹³C) domain of the spectrum.  ¹³C is broadband decoupled from the F2 (¹H) domain by applying a GARP pulse train (color coded in pink) at the ¹³C frequency during the collection of the FID.  One can turn each of these elements “on” or “off” for data collection.  The figure below shows the ¹H-¹³C gradient HSQC spectrum of benzene with all possible combinations of ¹H and/or ¹³C decoupling.

In the top left panel both ¹H and ¹³C decoupling are turned “on” and one observes a singlet in both the F2 (¹H) and F1 (¹³C) domains.  In the top right panel, the ¹H decoupling element is “on” while the ¹³C decoupling element is “off”.  The result is a ¹H-¹³C doublet in the F2 (¹H) domain and a singlet in the F1 (¹³C) domain.  In the bottom left panel, the ¹H decoupling element is “off” while the ¹³C decoupling element is “on”.  The result is a ¹³C-¹H doublet in the F1 (¹³C) domain and a singlet in the F2 (¹H) domain.  In the bottom right panel, both the ¹H and ¹³C decoupling elements are “off”.  The result is a ¹H-¹³C doublet in both the F2 (¹H) and F1 (¹³C) domains.

Calibration of 13C Decoupler Pulses

Proton-carbon HMQC, HSQC, and HMBC experiments rely on knowing the duration of the 90 degree pulse for ¹³C for at least one power level. These pulses must be known at both high power and low power when ¹³C decoupling is employed during the acquisition. This calibration is conveniently done with the pulse sequence depicted in the figure below for CHCl₃. For protons attached to ¹²C, the pulse sequence produces an out of phase signal. When no ¹³C pulse is applied (i.e. 0 degrees) the components of the ¹H-¹³C doublet signal (¹³C satellites) are antiphase with respect to one another. When a 90 degree ¹³C pulse is used, the ¹H-¹³C doublet is converted to an unobservable double quantum coherence and therefore is not present in the spectrum. When a 180 degree ¹³C pulse is employed, the ¹³C satellites will again be antiphase with one another but in the opposite sense compared to that when no ¹³C pulse was employed. The ¹³C decoupler 90 degree pulse is thus measured by collecting a series of spectra with increasing ¹³C pulse duration. The 90 degree pulse produces a null for the ¹³C satellites. This is depicted in the figure below. This must be carried out at both high and low ¹³C power to determine hard pulses to be used in pulse sequences and low power pulses to be used in multiple pulse decoupling schemes.

Searching for Small Peaks in 1H Spectra - 13C Decoupling

It is not usually necessary to decouple ¹³C while observing protons as ¹³C is only 1.1% naturally abundant and ¹²C has no spin. The only thing ¹³C decoupling will do for you is collapse the very small ¹³C satellites into single resonances which (aside from a very small isotope effect) will be coincident with the proton resonances for the protons bonded to ¹²C. In certain instances however, ¹³C decoupling is desirable. Such is the case when one is looking for very small peaks in a ¹H spectrum. Below is an example of how such small peaks can be observed more easily when the ¹³C satellites are removed with decoupling. The lower trace is a conventional proton spectrum while the upper trace is the same spectrum with ¹³C decoupling. This is very useful if one is searching for small amounts of impurities underneath the ¹³C satellites of much larger resonances.

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Why Are Some of My HMQC Correlations Doubled?

HMQC and HSQC signals are 1H detected with 13C decoupling. The sequences essentially throw away all of the signals for protons attached to 12C and retain all of those for protons attached to 13C. The coupling between 1H and 13C is used to establish a correlation but while the 1H signal is being detected, the 13C is decoupled. Occasionally you may find that some of your HMQC/HSQC correlations appear to be doubled or have a much lower than expected signal-to-noise ratio. Often this is due to poor 13C decoupling and is a problem for correlations at the extremes of the 13C chemical shift scale. At moderate magnetic field strengths, 13C "garp" decoupling is efficient over about 150 ppm or so. If the correlations are near the limits of the center of the 13C axis plus or minus (150 ppm/2), then you will find a loss in signal-to-noise ratio and perhaps some signal doubling due to partial 13C decoupling. Below is an example of the partial HMQC spectrum of 3-heptanone. In the left panel the 13C offset was set near the 13C resonances. In the right-hand panel the 13C offset was set at 110 ppm and the terminal methyl signal is poorly decoupled.

If you encounter this problem, center the 13C offset closer to the correlation which is poorly decoupled.