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.

Isotope Effects and the 19F - 13C HMQC Spectrum of Trifluoroacetic Acid

The ¹⁹F - ¹³C HMQC spectrum of trifluoroacetic acid is shown in the figure below.

The data were collected with a delay appropriate for a ¹⁹F - ¹³C J  coupling constant between the ¹J_F-C coupling constant of 284 Hz and the ²J_F-C coupling constant of 44 Hz.  The top and side traces are the one-pulse ¹⁹F and ¹³C spectra, respectively.  Why are the HMQC responses not at the same ¹⁹F chemical shift and why aren't they correlated to the peak in the ¹⁹F spectrum?  In order to answer these questions one must take into consideration the ¹⁹F - ^{12, 13}C isotope effects.  The chemical shift of the fluorine depends on whether it is bound to a ¹²C or a ¹³C.  The effect is largest across one bond and gets smaller over multiple bonds.  The ¹⁹F NMR spectrum for trifluoroacetic acid is shown in the figure below with and without ¹³C broadband decoupling in the upper and lower traces, respectively.

Approximately 98% of the trifluoroacetic acid is the ¹²CF₃-¹²COOH isotopomer, giving rise to a large singlet plotted off-scale in the figure. Approximately 1% of the signal is from the ¹³CF₃-¹²COOH isotoponer giving rise to a doublet with ¹J_F-C = 284 Hz and approximately 1% of the signal is from the ¹²CF₃-¹³COOH isotoponer giving rise to a doublet with ²J_F-C = 44 Hz.  All of these signals are clearly present in the lower trace of the figure.  When ¹³C broadband decoupling is applied, the doublets collapse into singlets.  The singlets from each of the isotopomers are resolved in the top trace.  The one-bond ¹⁹F - ^{12, 13}C isotope effect is 0.13 ppm and the two-bond effect is 0.02 ppm.  The figure below shows the same HMQC data with the spectrum from the top trace used as a projection.

One can see that the HMQC responses are correlated to their respective isotopomers.  These effects are also present in ¹H - ¹³C HMQC spectra, but the ¹H - ^{12, 13}C isotope effect is much smaller than the ¹⁹F - ^{12, 13}C isotope effect.  

19F - 13C HMQC

If one has an NMR spectrometer with hardware capable of synthesizing and amplifying the frequency of ¹⁹F from the ¹H channel and a broadband NMR probe whose ¹H channel can tune down to ¹⁹F, then one is able to do ¹⁹F - ¹³C HMQC experiments. The figure below shows an example of a ¹⁹F - ¹³C HMQC spectrum collected on a Bruker AVANCE 500 NMR spectrometer using a 5 mm broadband probe. The fluorine spectrum is plotted on the top and the ¹³C[¹⁹F] spectrum is plotted on the side. The panel on the left shows the spectrum optimized for one-bond coupling, while that on the right shows the spectrum optimized for two-bond coupling. Note that the protonated carbons are doublets as proton decoupling is not possible in this configuration. The large signal, off scale in the ¹³C[¹⁹F] spectrum is due to the solvent (benzene-d₆).

31P - 13C HMQC

When most people think about HMQC or HSQC spectra they think about protons and ¹³C or protons and ¹⁵N. Although these are by far the most common spins to probe, the HMQC technique can be applied to other spin pairs as well. In earlier posts to this BLOG, this was demonstrated for protons and ¹¹B and ³¹P and ¹⁰⁹Ag. This post shows an application of the technique to ³¹P and ¹³C. Measurements made between spin pairs, where one of the spins is not a proton, require a triple resonance probe and the appropriate filters.

The figure below shows ³¹P detected ³¹P - ¹³C HMQC data for dimethyl methylphosphonate with ¹³C decoupling during the acquisition and ¹H decoupling during the entire sequence. The spectra on the top and left-hand sides of the plots are separately run one dimensional ³¹P and ¹³C spectra, respectively, both with proton decoupling. The methyl carbon of dimethyl methylphosphonate has ¹J_CP = 142 Hz whereas the methoxy carbons have ²J_CP = 6 Hz. Two HMQC spectra were run. The one on the left was optimized for 142 Hz and the one on the right was optimized for 6 Hz. Each spectrum shows a correlation according to its coupling constant. The data were collected on a 500 MHz instrument with the appropriate bandpass filters on each channel.

Weak One-bond or Multiple Bond Correlations in 1H / 13C HMQC / HSQC Spectra

Many people are quite surprised to see either unusually weak one-bond correlations or weak multiple bond correlations in their ¹H / ¹³C HMQC / HSQC spectra. These people must be reminded that there is nothing "magic" about these experiments - the responses are based solely on an assigned delay proportional a reciprocal coupling constant. The large scale success of the ¹H / ¹³C HMQC / HSQC techniques can be attributed to the fact that most one-bond ¹H - ¹³C coupling constants are very similar ( ~ 145 Hz). The pulse sequences are therefore run with a delay based on a 145 Hz coupling constant. When one-bond coupling constants are significantly different than 145 Hz then the correlation will be either very weak or absent in the spectrum. Also, if multiple bond couplings are unusually large then those multiple bond correlations may be present in the spectrum. The figure below is an example. In the 500 MHz HMQC spectrum of an alkyne (optimized for 145 Hz coupling), one can see an unusually small one-bond correlation between the terminal alkyne proton and its attached carbon. There is also a weak two-bond correlation between the terminal alkyne proton and the other alkyne carbon.

HMQC vs HSQC

Proton detected Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Single Quantum Coherence (HSQC) are both NMR techniques used to correlate the chemical shift of the protons in a sample to a heteronucleus such as ¹³C or ¹⁵N via the J coupling interaction between the nuclei. Since both techniques essentially provide the same information - a correlation map between the coupled spins - students sometimes ask which of these two methods is better and which should they use routinely. The difference between the two techniques is that during the evolution time of an HMQC both proton and X magnetization (eg: X = ¹³C ) are allowed to evolve whereas in an HSQC only X magnetization is allowed to evolve. This means that an HMQC is affected by homonuclear proton J coupling during the evolution period while an HSQC is not affected as there is no proton magnetization during the evolution time. The homonuclear proton J coupling manifests itself as broadening in the X dimension. The top panel of the figure below shows the 7.05 T ¹H /¹³C HMQC and HSQC spectra of menthol with an expansion of one of the resonances highlighted in yellow. One can see that the expanded cross peak of the HMQC is broader in the ¹³C dimension than that of the HSQC. The bottom panel of the figure shows the corresponding ¹³C projection spectra. One can see that the resolution is better in the projection of the HSQC compared to the HMQC. One might conclude that, due to the higher ¹³C resolution, it is always better to run an HSQC rather than an HMQC. This is definitely the case if all of the pulses are calibrated well, however since there are many more pulses in an HSQC compared to an HMQC, it is more susceptible to losses in signal-to-noise-ratio due to poor probe tuning or poor pulse calibration. My advice to students is that, if high ¹³C resolution is required, then make sure the pulses are calibrated well on a well tuned and matched probe and run an HSQC. If high ¹³C resolution is not critical then run an HMQC.

1H - 11B HMQC

HMQC and HSQC experiments are very commonly applied to ¹H - ¹³C and ¹H - ¹⁵N. These techniques can also be applied to other isotopes. The figure below shows the ¹H - ¹¹B HMQC spectrum or ortho-carborane. The top and side traces are separately acquired ¹H [¹¹B] and ¹¹B [¹H] spectra, respectively. These data, combined with the ¹¹B COSY data, allow the complete assignment of the ¹H [¹¹B] spectrum.
Thank you to Dominique Duguay for providing the sample of ortho-carborane.

31P - 109Ag HMQC

Silver has two spin I = 1/2 isotopes (¹⁰⁹Ag and ¹⁰⁷Ag) with natural abundances of 48.18% and 51.82%, respectively. Although ¹⁰⁹Ag has a lower natural abundance, it is the preferred isotope to observe as its gyromagnetic ratio is slightly higher than that of ¹⁰⁷Ag. Direct observation of ¹⁰⁹Ag is problematic in that the Larmor frequency is low, the NOE's to protons are negative and most importantly, the T₁'s are very long (minutes to hours). In some silver phosphine complexes with ¹⁰⁹Ag - ³¹P coupling, the problems of observing silver directly can be circumvented by using indirect detection via a ³¹P detected ³¹P - ¹⁰⁹Ag HMQC. The advantages of using this approach are that the directly observed ³¹P frequency is 8.7 times higher than that of ¹⁰⁹Ag and the relaxation delay in the experiment depends on the ³¹P T₁'s which tend to be orders of magnitude shorter than those of ¹⁰⁹Ag.

Below is an example of a ³¹P detected ³¹P - ¹⁰⁹Ag HMQC for a silver phosphine complex. These data were acquired without ¹⁰⁹Ag decoupling. In the ³¹P trace on the top of the spectrum one can see a single ³¹P resonance with coupling to both ¹⁰⁹Ag and ¹⁰⁷Ag. The ¹⁰⁹Ag is indirectly observed in the F1 domain.

HMQC Responses in HMBC Data

Despite the built in low pass J filters written into the HMBC pulse sequences, the much larger one-bond 1H - 13C coupling responses are not fully suppressed. These one-bond HMQC responses show up as a pair of responses in the acquisition domain separated by the one-bond 1H - 13C coupling. The responses are doubled since 13C decoupling is not typically used in HMBC sequences. An example of these artifacts is shown below.

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.