2D-Heteronuclear : H-detected

HMQC
HSQC
HMBC
HMQC-TOCSY
HMQC-NOESY

A large number of pulse sequences have been proposed for "Reverse-Correlation" experiments which are based on the detection of proton spectra during t2 - the detection time - while the heteronuclear chemical shift is detected during t1 - the evolution time. This type of experiments have been proposed due to the increase of sensitivity that one can expect by detecting the most sensitive nuclei instead of the low-gamma nuclei like in HETCOR experiments (as a reminder, the sensitivity of a nuclei is proportional to the cube of its frequency!). In fact, when limited amount of material is available, the direct detection of Carbon-13 can be almost impossible but the detection of carbon chemical shift through those 2D "reverse" techniques is most of the time very easy. - I have seen cases where good HMQC could be obtained in 2 hr. but direct carbon gave very noisy spectra after 15 hr.

There are basically two types of experiments in this category: Those that utilize multiple quantum transitions during the evolution time (like HMQC, HMBC) and those using INEPT single quantum transitions during the evolution time (like HSQC)

HMQC (Heteronuclear Multiple Quantum Correlation)

The HMQC experiment provides correlation between protons and their attached heteronuclei through the heteronuclear scalar coupling. This sequence is very sensitive (compare to the older HETCOR) as it is based on proton detection (instead of the detection of the least sensitive low gamma heteronuclei).

The basic idea behind this experiment is in fact related to the echo difference technique, which is used to eliminate proton signals not coupled to the heteronuclei. (As a reminder, this cancellation is made possible by varying the phase of the pulses applied to the X nuclei (on alternate scans) and by subtracting the two signals - as can be seen in the figure below.

In this Spin Echo Difference experiment, a 180 degree (two 90` pulse with constructive phase: X, X) and no net pulse (two 90` pulse with destructive phase: X, -X) are applied on alternate scans. By subtracting the data in the receiver one cancel the unwanted protons attached to the unlabelled C-12. In order to get the 2D-shift correlation needed in HMQC, an evolution time t1 is introduced between the two heteronuclear 90` pulse.

• Therefore, the simplest version of the pulse sequence starts with a proton pulse followed by a 1/(2J) delay -During that delay, the proton magnetization (coupled to the heteronuclei) acquire anti-phase character.
• A 90` pulse applied to the X nuclei creates then multiple quantum magnetization which evolves under the influence of the X-chemical shift during the t1 evolution time (the proton chemical shift and heteronuclear coupling is refocused by using a 180` pulse at mid evolution time).
• At the end of the evolution period, another 90` pulse on the heteronuclei transfers the magnetization back into detectable (antiphase) single quantum proton magnetization which is then left to evolve another 1/(2J) delay to refocus the antiphased magnetization.
• The alternation in the phase of the last 90` heteronuclei pulse with the alternation of the receiver phase provides cancellation of the unwanted proton not coupled to the X-heteronuclei.
• During the proton detection period, the X-nuclei coupling interactions can be decoupled using Waltz or Garp decoupling sequences.

The minimum phase cycling require 4 scans in the classical phase sensitive experiment. By using pulse field gradients to select the coherence, it is now possible to run this experiment with only one scan! - This means that with a modern spectrometer, 2D-Shift correlation involving proton and carbon can be obtained in 5 minute!

HSQC (Heteronuclear Single Quantum Correlation)

The HSQC experiment have been proposed by Bodenhausen and Ruben (G. Bodenhausen and D. J. Ruben, Chem. Phys. Lett., 69, 185 (1980)). The HSQC experiment is in fact a double INEPT experiment. This experiment correlates protons with their directly attached heteronuclei. Proton magnetization is detected (during t2 - detection time) while the low-gamma nuclei evolves during the evolution time - t1. Because of the detection of the high frequency nuclei, this sequence is very sensitive. The enhancement in sensitivity this experiment permits is much greater than the enhancement obtainable by simple NOE (Nuclear Overhauser Effect). This is why this experiment have been referred to as the "OverBodenhausen" experiment.

• The HSQC experiment starts with proton magnetization. Therefore the recycle time is based on proton relaxation time (1.26 * T₁).
• The first INEPT step is used to create proton antiphase magnetization (2 delay) which is then transferred to the directly attached heteronuclei (Carbon, Nitrogen ...).
• This X nuclei magnetization is left to evolve with its chemical shift (during t1 - evolution time). The effect of proton coupling and chemical shift is removed by the use of a 180` proton pulse applied at mid evolution time.
• The double 90` pulse applied to both nuclei (in the beginning of the last INEPT step) transfers the magnetization back to proton as an anti-phased magnetization, which is then refocus during the last (2 delay). The proton in-phase magnetization can then be detected in the presence of the X-nuclei decoupler.

For further information, you can consult the literature for the comparison of the sensitivity of the HMQC and HSQC experiments.
A. Bax, M. Ikura, L.E. Kay, D.A. Torchia and R. Tschudin, J. Magn. Reson, 86, 304 (1990)
T.J. Norwood, J. Boyd, J.E. Heritage, N. Soffe and I.D. Campbell, J. Magn. Reson, 87, 488 (1990)

HMBC (Heteronuclear Multiple Bond Correlation)

The HMBC experiment detects long range coupling between proton and carbon (two or three bonds away) with great sensitivity. The length of the tau delay can be adjusted to detect relatively large coupling constants (4-10 Hz) tau = 0.06 s or smaller couplings (2-7 Hz) tau = 0.1 s.

In this sequence, the first 90` pulse on Carbon-13 serves as a low-pass filter that suppresses one-bond correlation and passes the smaller coupling. This pulse creates multiple quantum coherence for the one-bond coupling which is removed from the spectra by alternating the phase of the Carbon-13 pulse. The second 90` pulse on C-13 creates multiple quantum coherence for the long-range couplings. After the evolution time t1, the magnetization is converted back into detectable single quantum proton magnetization. The carbon decoupler is never used in this sequence: therefore the protons displays homonuclear as well as heteronuclear couplings.

This technique is very valuable to detect indirectly quaternary carbons coupled to protons - specially useful if direct Carbon-13 is impossible to obtain due to low amount of material available. This very useful sequence provide information about the skeleton of a molecule. It could be an alternative to the 2D-INADEQUATE experiment (which is so insensitive). It is also very useful in carbohydrate area as a sequence analysis tool that provides unique information concerning connectivities across glycosidic linkages. Another area of interest for using HMBC is in the peptide-protein area - specially when applied to a ^{15N labeled protein - It is possible with this technique to get connectivities between the Nitrogen and the CH proton of the amino acid of the next residue.}

HMQC-TOCSY (HMQC combined with a TOCSY experiment)

This sequence combines the power of HMQC and TOCSY together. In this experiment the carbon (or other heteronuclei) chemical shift is detected first during the evolution time. The magnetization is then transferred to its directly attached proton which is then correlated to the other protons in the same spin system during an MLEV mixing period prior to detection.

This experiment can be particularly useful in carbohydrate area as a tool that can spread the proton chemical shifts along the carbon axis. Indeed each carbons will correlate to its directly attached proton and to every proton that are part of the same sugar ring (the proton correlation depends on the length of the mixing period). Another application is in the peptide-protein area - when applied to ^{15N labeled protein - It is possible to get connectivities between the Nitrogen, the CH proton of the amino acid and the further away protons (depending on the length of the mixing time). The shift of the protons of each individual amino acid can be obtained that way (except for proline which do not have NH proton).}

There is also a version for HSQC-TOCSY.

HMQC-NOESY (HMQC combined with a NOESY experiment)

This sequence combines the power of HMQC and NOESY. In this experiment the carbon (or other heteronuclei) chemical shift is detected first during the evolution time. The magnetization is then transferred to its directly attached proton which is then correlated to the other protons that are close in space prior to detection.

This technique have found application in the peptide-protein area - when applied to ^{15N labeled protein - It is possible to get connectivities between the Nitrogen,} it's directly attached proton, and the protons that have NOE effect with it.

There is also a version for the HSQC-NOESY experiment.

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