4. Acquisition

Time requirement: 10 min Sample: 0.1 M ¹⁵N-labeled urea and 0.1 M '¹³C-labeled methanol in [D,]-DMSO.

Connect all three channels of the instrument for protein measurement as described in the introduction to this chapter. Insert the sample and wobble all three channels to opimum, beginning with ¹⁵N, then ¹³C, and finally ¹H, thus going from the lowest to the highest frequency. Determine the 90° ¹H pulse. Load the pulse program for the determination of a 180° selective ¹³C pulse. You have to set:

sw: 7 ppm o1: middle of ¹H NMR spectrum o2: 49 ppm for BC p1: 90° ¹H transmitter pulse [9 μs, –3 dB] p2: 180° ¹H transmitter pulse [18 ys, -3 dB] p3: 90° ¹³C decoupler pulse [20 μs, -3.3 dB] p4: 180° ¹³C shaped decoupler pulse [768 μs, g3 Gaussian cascade on 256 data points, attenuation to be varied, starting value 10 dB] dl:2s d2: 1/[2(C,H)] = 3.44 ms, calculated from 'I(C,H) = 145 Hz d3: p4/2= 384 μs d4: d2 – p4/2 = 3.056 ms rg: receiver gain for correct ADC input ns: 1 In contrast to Experiments 2.3, 2.5 or 7.3 this sequence yields an in-phase doublet.

Decrease the attenuation of the selective pulse in 1 dB steps and observe the variation of the signal strength. The 180° selective pulse is obtained when the methanol signals

5. Processing

Use standard 1D processing (see Exp. 3.1) applying an exponential window with a line-broadening factor lb = 0.3 Hz. Adjust the phase of the first spectrum for pure ab- sorption and always use the same phase correction.

6. Result

The figure shows the normal ¹H NMR spectrum of the sample obtained on a DRX-600 spectrometer with an inverse triple-resonance probe-head. For the determination of the pulses the methanol signals at 5= 3.15 are being used. The attenuation for the 180° pulse was obtained here as + 5.5 dB.