4. Acquisition

Time requirement: 2.3 h Sample: 3% strychnine in CDCl₃ Prior to the experiment the 90° pulse-length and transmitter attenuation for the spin-lock pulses must be calibrated (see Exp. 2.9). For optimum results on older instruments one should make allowance for the phase difference between the hard pulse p1 and the spin-lock pulses, either in the pulse program or in the adjustable parameter set if the software allows (see Exp. 7.1). Run a normal ¹H NMR spectrum of the sample and optimize the spectral width. Change to the 2D mode of the spectrometer software and load the ROESY pulse program. The duration of the spin-lock is an adjustable parameter. You have to set:

td2: 1 k data points in F2 td1: 256 data points in F; sw2: 10 ppm swl: 10 ppm o1: middle of ¹H NMR spectrum p!: 90° ¹H transmitter pulse p2, p3: series of 180° pulses at transmitter attenuation of spin-lock; 90° pulse- width and transmitter attenuation typically in the order of 90 xs and 23 dB. Total duration of spin-lock set to 300 ms by loop parameter n of spin-lock sequence. The loop parameter must be an even number, 832 was used initial value for ¢; evolution: 3 μs increment for ¢, evolution: 1/[2-sw1]

5. Processing

Apply zero-filling in F, to 512 real data points to obtain a symmetrical matrix of 512x512 real data points. Use an exponential window in F, and a squared 2/2-shifted sinusoidal window in F;. Apply real Fourier transformation corresponding to the TPP!

mode of data acquisition in F;. Phase correction in both dimensions is necessary. Adjust the phase of the diagonal signals negative so that the ROESY correlation signals are positive. TOCSY breakthrough signals would have the same phase as the

6. Result

The figure shows the result obtained on an AMX-500 spectrometer. Note that the diagonal signals have a negative phase (dotted contours). No TOCSY breakthrough