Relaxation delay

The remarkable stability and eontrollability of NMR speetrometers penults not only the preeise aeeiimulation of FIDs over several hours, but also the aequisition of long series of speetra differing only in some stepped variable sueh as an interpulse delay. A peak at any one ehemieal shift will typieally vary in intensity as this series is traversed. All the sinusoidal eomponents of this variation with time ean then be extraeted, by Fourier transfomiation of the variations. For example, suppose that the nomial ID NMR aequisition sequenee (relaxation delay, 90° pulse, eolleet FID) is replaeed by the 2D sequenee (relaxation delay, 90° pulse, delay i -90° pulse, eolleet FID) and that x is inereased linearly from a low value to ereate the seeond dimension. The polarization transfer proeess outlined in die previous seetion will then eause the peaks of one multiplet to be modulated in intensity, at the frequeneies of any other multiplet with whieh it shares a eoupling. 

The whole sequence of successive pulses is repeated n times, with the computer executing the pulses and adjusting automatically the values of the variable delays between the 180° and 90° pulses as well as the fixed relaxation delays between successive pulses. The intensities of the resulting signals are then plotted as a function of the pulse width. A series of stacked plots are obtained (Fig. 1.40), and the point at which the signals of any particular proton pass from negative amplitude to positive is determined. This zero transition time To will vary for different protons in a molecule. 

A number of parameters have to be chosen when recording 2D NMR spectra (a) the pulse sequence to be used, which depends on the experiment required to be conducted, (b) the pulse lengths and the delays in the pulse sequence, (c) the spectral widths SW, and SW2 to be used for Fj and Fi, (d) the number of data points or time increments that define t, and t-i, (e) the number of transients for each value of t, (f) the relaxation delay between each set of pulses that allows an equilibrium state to be reached, and (g) the number of preparatory dummy transients (DS) per FID required for the establishment of the steady state for each FID. Table 3.1 summarizes some important acquisition parameters for 2D NMR experiments. 

D, = relaxation delay (preparation period) t ,ta,t etc = fixed mixing periods NS == number of transients per FID DS = number of preparatory dummy transients per FID ... 

Dummy Scans and Relaxation Delays between Successive Pulses... 

To reach a steady state before data acquisition, a certain number of dummy scans are usually required. If the relaxation delay between the... 

Pre-saturation In this technique prior to data acquisition, a highly selective low-power rf pulse irradiates the solvent signals for 0.5 to 2 s to saturate them. No irradiation should occur during the data acquisition. This method relies on the phenomenon that nuclei which have equal populations in the ground and excited states are unable to relax and do not contribute to the FID after pulse irradiation. This is an effective pulse sequence of NOESY-type pre-saturation that consists of three 900 pulses RD - 900 - tx - 900 - tm - 90° - FID, where RD is the relaxation delay and t and tm are the presaturation times. 

Since the integration values form such an important element of structure determination, we need to set the spectrometer up properly before carrying out the NMR experiment. And one very important parameter which is often forgotten is the relaxation delay, the delay between the single NMR experiments which allows the nuclei to relax. Remember that relaxation is an exponential process, so that theory suggests that it is necessary for the best results to set this equal to at least five times (in our case more than 25 sec for the aromatic protons ). The other parameter we need to set correctly is of course the pulse angle, and the following set of experiments show how these are interrelated. 

We carried out two sets of experiments in which we set the pulse angle first at 90°, then at 30°. Using these two values we then varied the relaxation delay. Since the greatest difference in the relaxation times is that between the OH proton and the aromatic protons, we show in Fig. 11 the comparison between the integration values of the aromatic protons (set equal to 2.0) and of the OH proton for 90° pulses and for 30° pulses. The values approach each other with a relaxation delay of 10 sec and are virtually equal for a delay of 25 sec, but the 90° pulses give values which are completely wrong if a conventional delay of 1-2 sec is used On the other hand, the error is quite low if the delay is set at 2 sec and the pulse length is 30°. 

Fig. 11 Comparison between the integration values of the aromatic protons (set equal to 2) and of the OH proton for 90° pulses and 30° pulses as a function of the relaxation delay D1 in seconds...

Obviously we cannot however simply correlate the signal intensities with the presence of attached protons. So relaxation must also play a very important role. Relaxation times T, for carbon atoms also depend on whether these are protonated or not, and while T, for methyl or methylene groups may only be a few seconds, it may be as long as around 2 min for quaternary carbons Now the choice of an ideal relaxation delay becomes impossible, and so we have to make compromizes, which result in the large variations in signal intensity. 

However, in the inverse gated experiment it is very important that the relaxation delay chosen is very long, since the carbon atoms have very different relaxation times (and relax by different mechanisms). In our example the relaxation time was set to 120 seconds This of course makes the experiment a very time-consuming one (28 hours measurement time ). 

Fig. 35 Proton spectra obtained from the stopped-flow experiment. Above acetal 4. Below acetal 5. In each case 16 scans, relaxation delay 1 sec...

Fig. 37 Nitrogen-15 spectra of two aminophosphonates (structures as shown). 10-mm NMR tube, concentration 25% in CDC13, proton decoupling, relaxation delay 15 sec, measurement time 12 hours...

In Problem 49 we recorded the carbon-13 spectrum using a relaxation delay of 25 sec with the shorter delays we tend to use routinely the signals due to the quaternary carbons would have been almost invisible ... 

Long relaxation delay (25 sec) increases intensities of the quartemary C atoms ... 

Figure 27 Edited broadband HMBC spectrum of cyclosporine using the pulse sequences shown in Figure 26 in an interleaved manner. The two subspectra, CH + CH3 (left) and C + CH2 (right), exemplify the editing properties. The spectrum in the bottom displays the two subspectra, CH + CH3 (black) and C + CH2 (grey) in the same frame. The number of scans was 32 for each of the 128fi increments, the relaxation delay was 1 s, and the range for the third-order low-pass. /-filter was 115 Hz < Vch < 165 Hz. The spectra were processed to maintain the absorptive profiles in F, while a magnitude mode was done in F2.

Shorten or even eliminate the relaxation delay is only effective for relatively fast-relaxing nuclei, and can lead, if decoupling is turned on, to serious radio-frequency heating problems if the relaxation delay is very short. Recently, Kupce and Freeman have introduced an alternative... 

The essence of the ASAP method is based on the different spin-lattice relaxation behaviour of these two types of protons. In the case of a HMBC experiment, the acceptor protons are those directly bound to 13C with large spin-spin couplings they are the spins that give rise to the final spectrum. By contrast, the donor protons have negligible couplings to 13C and are therefore essentially unaffected by this polarization transfer sequence, which simply returns them to the z axis. In their method, Kupce and Freeman have proposed to replace the usual relaxation delay with a short cross-polarization (HOHAHA) interval. This offers a... 

All preparations were structurally characterized by means of XRD (Siemens 5005). TEM imaging was performed with a Philips CM200 instrument. 27A1 and 29Si MAS NMR (Broker 500 MFlz and 360 MFlz respectively) was used to study the microporous phase and the kinetic of its formation. The relaxation delays were 0.2s and 200s respectively. Acidity was determined by the adsorption of carbon monoxide after activating the samples in vacuum (10 6 mbar) at 450°C for 1 h. The spectra were recorded on a Equinox 55 Broker spectrometer with a resolution of 2 cm 1 and normalized to 10 mg of sample. 

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