Coherence selection

Figure 13 Timing diagram for the clean HMBC experiment with an initial second-order and terminal adiabatic low-pass 7-filter.42,43 The recommended delays for the filters are the same than for a third-order low-pass J filter. <5 and 8 are gradient delays, where 8 — <5 + accounts for the delay of the first point in the 13C dimension. The integral over each gradient pulse G, is H/2yc times the integral over gradient G2 in order to achieve coherence selection. The recommended phase cycle is c/)n = x, x, x, x 3 — 4(x), 4(y), 4( x), 4(—y) with the receiver phase c/)REC = x, x.

The exploitation of the above expression systems in FRET requires the coherent selection of donor and acceptor moieties, from both the spectroscopic and biological perspective relative expression levels, compartmentalization, and temporal evolution of the system under study. Very advantageous are combinations of small fluorophores with VFPs, as well as with fluorescent nanoparticles, particularly QDs. 

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. 

Coherence selection using pulsed field gradients... 

Aliasing in the remote dimension is truly worthwhile only if the sample concentration is high enough to collect only the minimum number of scans per increment required for proper coherence selection in other words, the acquisition is not S/N limited. If so, aliasing offers a proportional saving in acquisition time and data size for a given spectral resolution. 

With the carrier off-resonance coherence selection may be completely omitted, too, because most SQ and various MQ cross peaks will appear shifted from each other in the remote dimension. The separation is a function of the distance between the individual (SQ) resonances and the carrier. All number of quanta, including OQ correlations, can be covered by removing the refocusing 180° pulse in the middle of the MQ excitation delay. This introduces modulation of cross peak intensities by the chemical shift, too, however [6]. 

Figure 3 presents an example of such a situation. The 2Q-HoMQC spectrum of apo-cytochrome c was acquired in 93% H2O at 480 ixM concentration on a Varian Unity/INOVA 600 MHz NMR instrument overnight, using a pulse sequence with gradient coherence selection and weak gradient spin-echo during excitation delays and the evolution period [29], respectively (I.P., not published). The spectral windows were 8 kHz both in F2 and F. ... 

Fig. 4. Modified X/Y IMPEACH-MBC pulse sequence used for 19F/15N shift correlation according to Ref. 27. The notation of 90° and 180° pulses is as before. The (d/2 — 180°(Y) — d/2) element represents a variable delay that is incremented concurrently with the decrementation of the accordion delay vd. Pulse phases are x, unless specified x = — x 2 = x, — x 3 = x, x, — x, — x = , — x, — x, x. The bipolar gradients Gs flanking the 180°(Y) pulse can be set to arbitrary power levels, and the relative strengths of the coherence selection gradients G and G2 are determined by G2/G1 =2 Yy/Tx-...

X/Y coherence transfer steps as in the original HNCA experiment.41 As in the previous case, X nuclei are selectively irradiated by low power rectangular or shaped pulses, and coherence selection is accomplished by the matched pulsed field gradients Gb G2 and further assisted by a spoil gradient Gs. Owing to the need to avoid -pulses in the X-channel, the spectra are processed in magnitude mode in the fft dimension. 

The idea of back transformation of a three-dimensional NMR experiment involving heteronuclear 3H/X/Y out-and-back coherence transfer can in principle be carried to the extreme by fixing the mixing time in both indirect domains. Even if one-dimensional experiments of this kind fall short of providing any information on heteronuclear chemical shifts, they may still serve to obtain isotope-filtered 3H NMR spectra. A potential application of this technique is the detection of appropriately labelled metabolites in metabolism studies, and a one dimensional variant of the double INEPT 111/X/Y sequence has in fact been applied to pharmacokinetics studies of doubly 13C, 15N labelled metabolites.46 Even if the pulse scheme relied exclusively on phase-cycling for coherence selection, a suppression of matrix signals by a factor of 104 proved feasible, and it is easily conceivable that the performance can still be improved by the application of pulsed field gradients. 

Phase cycling and magnetic field gradients for coherence selection... 

The spectral quality and the efficiency of the basic COSY and the DQ-filtered COSY experiments may be improved with the use of field gradients instead of phase cycling for coherence selection, which remove spectral artifacts and make time consuming phasecycling superfluous. 

A variety of sequences exist, which differ with respect to the detected interaction ( J, or Jx ) and the mode of detection ( C or H detected, magnitude or phased mode, phase cycling or gradients for coherence selection). In view of the reduced sensitivity of heteronudear experiments with respect to homonuclear COSY experiments and the steadily decreasing sample amounts submitted for NMR experiments, there is no doubt that the inverse ( H) detected, gradient enhanced experiments are currently the best methods to apply. However on older type spectrometers, not equipped for inverse detection the old-fashioned direct C detected experiments are still in use. 

Mao, J. D., and Schmidt-Rohr, K. (2005). Methylene spectral editing in solid-state l3C NMR by three-spin coherence selection. J. Magn. Reson. 176,1-6. 

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