Delayed protons 5-value

Because both spins are in the transverse plane and transition energy levels are matched, energy can be transferred from the protons to the nuclei. In this manner the rate of repolarization is controlled by rather than by Because the protons can interchange energy by spin-diffusion only a single-proton exists and its value is usually on the order of 1 s. As a result the preparation delay can be reduced from 10 s to about 5 s increasing the number of transients, which can be acquired by two or more orders of magnitude. 

It is important to avoid saturation of the signal during pulse width calibration. The Bloch equations predict that a delay of 5 1] will be required for complete restoration to the equilibrium state. It is therefore advisable to determine the 1] values an approximate determination may be made quickly by using the inversion-recovery sequence (see next paragraph). The protons of the sample on which the pulse widths are being determined should have relaxation times of less than a second, to avoid unnecessary delays in pulse width calibration. If the sample has protons with longer relaxation times, then it may be advisable to add a small quantity of a relaxation reagent, such as Cr(acac) or Gkl(FOD)3, to induce the nuclei to relax more quickly. 

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. 

The INEPT experiment can be modified to allow the antiphase magnetization to be precessed for a further time period so that it comes into phase before data acquisition. The pulse sequence for the refocused INEPT experiment (Pegg et al., 1981b) is shown in Fig. 2.13. Another delay, A. is introduced and 180° pulses applied at the center of this delay simultaneously to both the H and the C nuclei. Decoupling during data acquisition allows the carbons to be recorded as singlets. The value of Z), is adjusted to enable the desired type of carbon atoms to be recorded. Thus, with D, set at V4J, the CH carbons are recorded at VsJ, the CH2 carbons are recorded and at VeJ, all protonated carbons are recorded. With D3 at %J, the CH and CH ( carbons appear out of phase from the CH2 carbons. 

We can see at once that each proton behaves differently, because it has its individual relaxation time Tx depending on the delay signals may be negative, positive, or have zero intensity. The T, values can be computed using spectrometer software. 

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...

In addition to measuring TCH for the polymorphic system in question, the proton T value must be determined since the repetition rate of a CP experiment is dependent upon the recovery of the proton magnetization. Common convention states that a delay time between successive pulses of 1-5 X T, must be used. Figure 10B outlines the pulse sequence for measuring the proton Tx through the carbon intensity. One advantage to solids NMR work is that a common proton Tx value will be measured, since protons communicate through a spin-diffusion process. An example of spectral results obtained from this pulse sequence is displayed in Fig. 12. 

Fig. 5 Effect of varying relaxation delays between on- and off-resonance experiments in STD NMR experiments, a Experimental setnp for interleaved measnrements in STD NMR spectroscopy, n represents the nnmber of scans. The inter-scan delay Adi is varied while keeping on- and off-resonance freqnencies constant at -4 and -t300 ppm, respectively, b The resulting STD effects for the 0-methyl group of a-L-Fuc-O-methyl in the presence of RHDV VLPs. The equation that was used for non-linear least squares data fitting is based on the saturation recovery experiment [98], With Ti(iig) = 0.91 s as measured independently (unpublished results) and a Monte Carlo error estimation yields Ti(virus) = 10.06 0.41 s. This value does not directly correspond to a Tl relaxation time of the virus protons, because other factors also influence the observed relaxation [99]. According to these findings a relaxation delay Adi = 25 s was employed in all STD experiments. This results in a recovery of 92% of the virus resonance, and thereby reduces errors in epitope mapping that are introduced otherwise by non-homogeneous recovery of the binding site.

NOE Measurements. The one-dimensional NOE data were collected at 300 MHz on a Bruker AM-300 NMR spectrometer operating at 300 K. Because the relaxation times for the protons of the hexa-deuterated compound ranged from O.A s to 3.8 s, delays of 20 s. were used between scans. Values for the T s were also measured and found to range from 0.32 s to 1.5 s all values were consistent with a rotational correlation time of 1.1x10 s. 

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