Gradient-selected HSQC

Gradient-Selected HSQC with Phase-Sensitive Data Presentation... 

Figure 6.11. A gradient-selected HSQC spectrum of the carbopeptoid 6.3 at natural N abundance plotted at high and at low contour levels to show the thermal noise floor. No ti-noise artefacts remain from the parent resonances ( N is referenced to external liquid ammonia).

A further improvement is the gradient selected HSQC experiment that can be run like the gradient selected HMQC experiment with one scan per experiment. In the following Check it an experiment with decoupling during acquisition is simulated and compared with the other HSQC experiments which differ in the detection mode and the sensitivity enhancement. 

Load the configuration file ch722b.cfg and run a gradient selected HSQC experiment with TPPI detection. Compare the result with the similar HSQC experiment under echo/antiecho (E/A) detection, i.e. with the same number of scans and same experimental parameters. Extract the rows containing the correlation peaks. 

Figure 1 shows the five basic independent steps that can be identified in a standard 2D gradient-selected HSQC pulse scheme. Step i The pre-scan period is usually defined by a long recycle delay (some seconds of duration, accordingly to the existing Tj( H) relaxation times) to allow the recovery of the magnetization to a pre-equilibrium state just before... 

Fig. 2. Pulse scheme for the gradient-selected, sensitivity-enhanced X/Y se-HSQC experiment as employed for 31P/15N correlation spectroscopy in Ref. 25. 90° and 180° hard pulses are denoted by solid and open bars, respectively. 2 are delays of length 1/(4 /x,y)> and is a short delay of the same length as the gradient pulse (typically rj 1 ms). Pulse phases are x, unless specified. The ratio of gradient pulse strengths is set to G2/Gi = Yy/Yx, and quadrature detection in Fi is achieved by recording every transient twice and changing the sign of G2 in the second scan.

Gradient-selected HMQC/HSQC with GARP decoupling, see discussion earlier. 

The assignment of the carbohydrate chains of an intact glycoprotein (de Beer et al., 1994) with the help of a gradient-selected natural abundance HSQC-TOCSY spectrum is a recent example of the use of Hartmann-Hahn-type experiments in the assignment of oligosaccharides (Dabrowski, 1994). 

So great are the advantages of the so-called inverse-detected experiments that little use is now made of the older normal-mode experiments, except on older instruments. The former acquires data in the proton dimension, while the latter the carbon dimension. Satisfactory HMQC (or HSQC) spectra can be run in as little as 4 minutes with gradient selection [2], or about 15 minutes without. Longer experiments are still recommended for detailed work, since longer acquisitions obviously still give valuable signal-to-noise improvements and consequent interpretability enhancements. 

Figure 6.10. A gradient-selected, phase-sensitive HSQC sequence using the echo-antiecho approach. The N- and P-type pathways are selected by the last gradient.

A similar logic to that above applies to gradient selection in the HSQC experiment, for which a variety of different approaches are also possible [7]. A suitable sequence employing the echo-antiecho approach is illustrated in Fig. 6.10, and requires only two gradients in proportion to the magnetogyric ratios of the X and H spins since each acts on single-quantum X and H magnetisation. Thus, for a correlation experiment, ratios of 4 1 and... 

Figure 6.18. The gradient-selected, spin-echo HSQC sequence for multiplicity editing within the 2D correlation experiment. Setting A = 1/2J inverts XH2 responses relative to those of XH and XH3.

HSQC pulse sequence with gradient selection. The CTP for an N-type spectrum is shown by the full line and for the P-type spectrum by the dashed line. 

In the heteronuclear experiment category, the experiments of interest are the heteronuclear multiple quantum correlation (HMQC) experiment, the heteronuclear single quantum correlation (HSQC) experiment, and the heteronuclear multiple bond correlation (HMBC, including the gradient-selected version gHMBC) experiment. Both the HMQC and HSQC produce similar results, but each has its own unique advantages and disadvantages. 

Fig. 14.51 (A) HSQC spectrum of a 1-mM spectrometer using the gradient selected sen-...

The pulse sequence resembles superficially that of the sensitivity enhanced HSQC [462]. Obviously neither proton during nor N during the acquisition f2 should be decoupled to maintain the multiplet components. It is the latter part of the pulse sequence used for the reverse transfer from to including the gradient selection that chooses the most slowly relaxing multiplet component for the detection (Fig. 14.52). 

Fig. 3. Sensitivity obtainable in high-pressure borosilicate glass cells and sapphire eells. The sample contained 0.5 mM uniformly N-enriched Csp from Thermotoga maritima (TwCsp) in 50mM phosphate buffer (pH 6.5), 20mM NaCl, 0.2mM Na-EDTA, 0.1 pM NaNj, 10% D2O and 90% H20. Gradient selected sensitivity enhanced H- N-HSQC spectra were recorded under identical experimental conditions either in a sapphire cell (left) with an outer diameter of 3.18 mm, inner diameter of 1.72 mm or a borosilicate glass capillary (right) with an outer diameter of 5.0 mm and an inner diameter of 1.2 mm. Data were recorded with a 8 mm inverse triple-resonance probe at 600 MHz proton frequency. Total acquisition time, approximately 2.5 h resolution, 2048 points in the direct dimension and 256 points in the indirect dimension. The temperature was adjusted to 303 K. (Top) Only a small part of the spectra are shown and plotted at the same contour levels for the two experiments. (Bottom) 1-D trace through the maximum of the H -signal of K19. (After ref. 35.)...

Baiagern et al. [8] assigned hydrolyzed poly(TEE-co-PSEPVE) chemical shifts through characterization of the prepolymer, the sulfonyl fluoride form of Nafion perfluoropolymer membrane (12). It was assumed that the chemical shifts of the prepolymer would closely match those of the final polymer since the structures are nearly identical. The perfluorinated Nafion prepolymer was characterized with i F-i F selective COSY and gradient crisis -HSQC (gc2HSQC). 

The 15N,1H shift correlation maps are most conveniently recorded with a sensitivity-enhanced HSQC sequence with incorporated water flip-back pulses for reduced saturation transfer and pulsed-field gradients for coherence selection. The pulse sequence of the experiment is shown in Fig. 14.4 A. 

Fig. 14.4 Pulse sequences used for the experiments described in this chapter. A [ N HJ-HSQC with water flip back and PFGs. The shaped pulse on the proton channel is a sine-shaped, 1.5 ms soft pulse all other pulses are hard pulses. Gradients are applied as square or sine-shaped pulses. The sign of the last gradient is reversed for anti-echo selection together with the sign of phase 6. B CPMG sequence. C bpPFGLED sequence. The delay T denotes the diffusion delay. Typically, r is set to 1 ms, T to 50-100 ms and Te to 1.2 ms.

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