Pulse sequence modified INEPT

The INEPT experiment can be modified to allow the antiphase magnetization to be precessed for a further time period so that it comes into phase before data acquisition. The pulse sequence for the refocused INEPT experiment (Pegg et al., 1981b) is shown in Fig. 2.13. Another delay, A. is introduced and 180° pulses applied at the center of this delay simultaneously to both the H and the C nuclei. Decoupling during data acquisition allows the carbons to be recorded as singlets. The value of Z), is adjusted to enable the desired type of carbon atoms to be recorded. Thus, with D, set at V4J, the CH carbons are recorded at VsJ, the CH2 carbons are recorded and at VeJ, all protonated carbons are recorded. With D3 at %J, the CH and CH ( carbons appear out of phase from the CH2 carbons. 

Several modified INEPT and DEPT pulse sequences have recently been introduced (IS) (see Fig. 1). The new INEPT+, DEPT +, and DEPT + + sequences differ from the original INEPT and DEPT sequences only in that they employ additional refocusing and purging pulses. These serve to reduce or eliminate distortions inherent in the parent pulse sequences. The fundamental polarization transfer mechanism however remains unchanged. 

NOESY-HMQC spectrum for a uniformly deuterated C-labelled protein with protonated Me-groups. The HMQC sequence utilises methyl-TROSY evolution, reducing the relaxation rates, allowing to extend the acquisition times. The modified pulse sequence of the experiment was presented that makes use of the INEPT period to record evolution. The experiment took 6.5 days to measure, that constituted 30% of the experiment time based on the uniform data sampling, resulting in a 4D spectrum with a resolution similar to that of a 3D spectrum. The data were processed using multidimensional decomposition. Both of the above processing methods produce data formats compatible with currently used spectra analysis software. 

The sequence can easily be modified to allow the two separate components to combine—refocused INEPT. After the final 90° pulses but before data acquisition, a period 2A is added, with A = 1/4J, during which the two vectors combine. H and 13C 180° pulses are placed in the middle of this precession period to refocus chemical shifts. If decoupling is now applied during data acquisition, a single line of intensity 8 is obtained, that is, four times as large as would be obtained... 

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