4. Acquisition

Time requirement: 5h Sample: 3% strychnine in CDCl}.

Record normal 1D ¹H and ¹³C NMR spectra, optimize the spectral widths and note the offsets. Switch to the 3D mode of the spectrometer software and load the 3D-HMBC pulse program. You have to set:

td3: 512 data points in F; (¹H) td2: 256 data points in F, (¹³C) td1: 16 data points in F\ (J(C,H)) sw3: 9.5 ppm sw2: 187 ppm swl: 250 Hz o1: middle of ¹H NMR spectrum o2: middle of ¹³C NMR spectrum p1: 90° ¹H transmitter pulse p2: 180° ¹H transmitter pulse p3, p4, p5: 90° ¹³C decoupler pulse dl:2s d2: 1/[2(C,H)] = 3.5 ms, calculated from 'J(C,H) = 145 Hz g1, g2, g3: sinusoidal-shaped field gradients with 5% truncation, | ms dura- tion and ca. 0.1 T/m strength, with gradient loop counters, ring-down delays (100 μs), lock blanking and gradient coil blanking switches according to actual instrumentation used. Gradient strength ratio: 2:2: 1 initial value for t; evolution: 20 ms initial value for f, evolution: 3 μs increment for ¢, evolution: 1/sw1; increment for ¢, evolution: 1/[2-sw2]

5. Processing

Apply zero-filling to 512 real data points in F and to 32 real data points in F; to ob- tain a matrix of 256x512x32 real data points. This will result in 4 MB of processed data. Use a n/2-shifted squared sinusoidal window in all three dimensions. Apply the correct acquisition order parameter (3-1-2) before FT. Phase correction is not neces- sary, since a magnitude calculation is performed after the last Fourier transformation in F,. Further details are very dependent on the particular software you use to process such a 3D data file. Note that the 3D cuboid obtained is not further inspected; instead, the F,-F projection is calculated and displayed.