g1–g11: sinusoidal-shaped field gradients, ca. 0.1 T/m strength, with gradient loop counters, ring-down delays (50 μs), lock blanking and gradient coil blanking switches according to actual instrumentation used. Gradient duration and strength according to the table:

gradient	g1	g2	g3	g4	g5	g6	g7	g8	g9	g10	g11
duration [ms]	1	1	t₁/2	t₁/2	0.5	0.5	1	1	1	1	0.5
strength [%]	30	30	3	3	80	80	45	45	50	50	16.2

increment for t₁: 1/[2·sw1]
ds: 16
ns: 16

5. Processing

Apply zero-filling to 512 real data points in F, to obtain a matrix of 512x512 real data points. Use Gaussian multiplication [gb = 0.1, lb = –2 Hz] in F2 and 7/3-shifted squared sinusoidal window in F). Phase correction and base-line correction may be necessary for both dimensions. Reference the ¹⁵N dimension using the =-scale procedure described in the introduction to this chapter.

6. Result

The figure shows the 2D spectrum obtained on a DRX-600 spectrometer with a multinuclear inverse z-gradient triple-resonance probe-head (fixed third channel on “C). Although ubiquitin is too small a protein, and the instrument used has too low a magnetic field to exploit the advantages of the method efficiently, the displayed spectrum shows the successful application of the principle. Note that all signal positions are off- set by ca. 45 Hz in both dimensions, which is half of the N,H spin coupling constant. If one wants to compare the result with other methods this should be taken into ac- count during the referencing procedure. Note, furthermore, that compared with the figure of Experiment 15.2, all NH2 signals of the side-chains are missing.

7. Comments

The pulse sequence is divided into four sections a–d separated by dotted vertical lines. First we find in a a standard INEPT transfer from proton to ¹⁵N yielding the coherence 2/y,/N, after pulse p10 as described for the standard HSQC experiment 10.17. The two gradient pulses g1 and g2 correct for non-ideal 180° pulses.

In the next section b chemical shifts of ¹⁵N are sampled. A special feature not yet shown in this book are the weak gradient pulses g3 and g4 which change their sign in the middle of the f, period. They suppress effects of radiation damping during f,.

At the end of the ¢; period the two gradients g5 and g6, which are separated by a 180° ¹⁵N pulse p11, encode in an echo/anti-echo manner the signals of protons that are bonded to ¹⁵N.