The ge-2D HSQC experiment is the gradient-enhanced version of the conventional 2D HSQC experiment in which coherence selection is achieved by means of PFG. Thus, clean 2D HSQC spectra can be recorded in a single scan per t₁ increment without need for phase cycle when sample concentration is high. Other advantages are the optimal dynamic range, improved water and artefact suppression, and reduced t₁ noise in the minimally required experiment time. The HSQC experiment allows to trace out directly bonded ¹H-X pairs via the large ¹J_HX coupling constant
 

Easy implementation on any AVANCE spectrometer equipped with pulsed field gradients (PFGs) and inverse probehead.

In addition of the advantages to use PFGs, an important aspect when PFG are incorporated in the HSQC pulse sequence, are the sensitivity and resolution requirements. The effect on the sensitivity of several gradient-based HSQC and other related schemes have been extensively discussed ( 95JB11-6 , 95JMRB235-108 , 94JMRA70-111 , 96JMRA64-122 , 94JB301 , 93ANG1489 , and 96JMRA17-1191 ). It is possible, for instance, to design six different basic versions of the 2D ¹H-X HSQC experiments using PFGs. The use of each version will depend on the sample under study.
 

• The dephasing gradient G1 is applied into the variable t₁ period, obtaining a magnitude mode spectrum ( 91JACS9688 and 92JMR282-100 ). This is an excellent option for routine samples in which sensitivity and resolution are not critical. In this case, two gradients with a  4:1 ratio select N-type data (shaded line corresponding to a p₁=+1) whereas a 4:-1 ratio would select P-type data (continuous line corresponding to a p₁=-1).

• The echo-antiecho version of this experiment uses the same sequence, but the intensity of the refocusing gradient G₂ is inverted on alternated scans to obtain the N- and P-type data separately ( 92JMR207-98 and 92JMR660-98 ). After proper processing, this approach allows for obtain phase-sensitive spectra but with sensitivity losses by a factor of square(2) with respect to the classical phase-cycled experiment.

• Use of the PEP methodology ( 91JMR429-91 , 91JMR151-93 , and 93AR1 ). This is the best option to record phase-sensitive 2D HSQC spectra with maximum sensitivity ( 92JACS10663 ). The selection procedure use the same principles described for the echo-antiecho approach, but the pulse sequence must be modified adding a second retro-INEPT block in order to select both orthogonal components of the magnetization (I_zS_x and I_zS_y) present during t₁. This basic scheme is widely applied to improve the sensitivity in other related multidimensional experiments ( 95JB11-6 , 95JMRB235-108 , 94JB301 , 93ANG1489 , and 96JMRA171-119 ). In triple resonance experiments applied to labeled proteins, sensitivity can be further enhanced by simultaneous acquisition ( 95JB97 ). Some examples of modified PEP-HSQC experiments have been described to measure accurate 1JNH ( 96JMRB245-112 and 96JMRB269-112 ), measure a set of relaxation parameters in ¹⁵N-¹H spin systems ( 96JMRB245-112-111 and 96JMRB269-101 ) or to observe exchange broadened signals ( 96JB223 ).

• A X filter-z (consisting of 90°(X)-PFG-90°(X)) is applied between the t₁ period and the dephasing G₁ gradient ( 93MRC287 ). A phase-sensitive spectrum is obtained using the classical acquisition and processing procedures (for instance, TPPI), but theoretical sensitivity loss by a factor of two is obtained compared to the phase-cycled experiment. However, this loss can be partially recovered applying the PEP methodology (a second retro-INEPT block shifted 90º in phase) without the need to apply the echo-antiecho approach ( 95JMRB285-108 ).

• Use of PFG as purge elements in the original phase cycle sequence. These PFG are placed between the simultaneous 90° pulses of ¹H and X in order to select the corresponding IzSz magnetization ( 93JMRA113-101 and 93JMRB239-102 ). Although this is not a pure selection procedure, this approach reduces the number of phase cycle steps and minimizes the suppression artefacts without affecting the overall sensitivity with comparison to the phase cycled analog.

• Much attention has been given to the effect of water suppression on the signal intensity of relatively rapidly exchanging protons, as the NH protons in proteins. As a general approach, the WATERGATE block ( 92JB661 and 93JMRA241-102 ) is usually applied during the retro-INEPT block of the standard HSQC pulse sequence in order to improve solvent suppression in aqueous samples. WATERGATE can also be combined with the water flip-back approach ( 93JACS12593 ) which ensures that the water magnetization is little perturbed and oriented along the +z axis during most of the experiment, especially just prior to acquisition, to minimize the saturation of water. This is commonly applied to proteins and nucleic acids dissolved in H₂O. Other related approaches have been proposed to avoid the saturation transfer from water in HSQC experiments ( 93JMRB315-101 , 94JMRA178-107 , 94JMRB45-105 , 95JMRB94-108 , and 96JACS5510 ).

Other related versions:

• Simultaneous acquisition of ¹H-¹³C and ¹H-¹⁵N 2D HSQC spectra can be obtained using the Time-Shared method
• Incorporation of an X-filter prior to acquisition to remove ABX strong coupling signals ( 99JMR89-138 )
• Improved resolution in the F₁ dimension can be achieved by applying a band-selective carbon pulse ((see Semi-selective HSQC experiment). This approach can be applied in any version of the mentioned HSQC experiment.

The ge-2D HSQC experiment is usually recorded in routine and automation modes. Minor changes are required if a predefined parameter set is available. The user must define the strength, the duration, and the shape of the gradients and the recovery delay.

More details on practical implementation of ge-2D HSQC experiments on AVANCE spectrometers can be found in the corresponding tutorials:

Tutorials: 2D inverse experiments

Tutorials: 2D gradient-based inverse experiments

The HSQC spectrum correlates chemical shifts of heteronucleus X (F₂ dimension) and protons (F₁ dimension) via the direct heteronuclear coupling ¹J(XH). The effective suppression of unwanted ¹H-¹²C or ¹H-¹⁴N magnetization by means of PFGs allows to obtain ultra-clean 2D spectra from which clear analysis can be done.

Related experiments:

2D Inverse experiments

2D Inverse gradient-enhanced experiments