Redor RELOAD

As DCP did not yield gains in signal to noise, when adapted for RELOAD enhance-ment, the Redor sequence was considered. As was seen before for the RELOAD-PDSD experiments, shaped pulses alone seem to be minimally perturbing to the bath mag-netization, in contrast to the spin lock field used during DCP. Therefore it should be possible to achieve significant RELOAD enhancement, if only shaped pulses are used in context of the correlation experiment. Thus the Redor sequence is an ideal candidate.

In Fig. 3.41 b) the RELOAD adaption of the Redor sequence is shown. Here, all

3.6. APPLICATION OF RELOAD TO HETERO-NUCLEAR CORRELATION SPECTRA

Figure 3.40: Spectra showing the effect of an increase of the second spin lock time from 3 msec to 5msec in an¹H−¹³C−¹⁵N DCP experiment. A significant decrease in¹³Cmagnetization remaining after the¹³C−¹⁵N contact can be observed, without an accompanying rise in polarization on the¹⁵N channel (not shown). This suggests a perturbing effect of the spin lock field on the ¹³C magnetization. No RELOAD was used for either spectrum. Both spectra were recorded with at 600 MHz, 10 kHz spin rate, 295K and 72 scans. A Q5 Gaussian cascade of 5 msec length was used for selective Cαexcitation at -3500 Hz off resonance (which was set to 75ppm). 20 Hz exponential line broadening was applied to all spectra.

Figure 3.41: Comparison of a conventional Redor sequence and its adaption to RELOAD. In a) a conventional Redor sequence is shown, for sake of comparison, with its RELOAD version shown in b). All pulses on the¹³C channel (save for the pulse prior to the first z-filter) have been replaced by selective pulses. After acquisi-tion, a nested inner loop is performed. To investigate the effect of multiple shaped pulses on the bath magnetization, sequence b) was modified as shown in c). Here, the RELOAD cycle commences before the acquisition. After the last RELOAD loop the 85

3.6. APPLICATION OF RELOAD TO HETERO-NUCLEAR CORRELATION SPECTRA

Figure 3.42: Overlay of aCO band selective Redor spectrum, recorded with the se-quence shown in Fig. 3.41 b) without RELOAD (blue) and with three RELOAD cycles (red). The signal to noise ratio is significantly enhanced, without any perturbation of the peak shape. The spectra have been scaled to the same level of noise. Both spectra were recorded with at 600 MHz, 8 kHz spin rate, 295K and 128 increments in the indi-rect dimension and 120 scans. Sinc pulses of 250 usec were used with different power levels for selectiveCOexcitation and inversion, 11000 Hz off resonance (which was set to 100ppm). 50 Hz exponential line broadening was applied in both dimensions.

pulses on the¹³Cchannel (save for the pulse prior to the first z-filter) were replaced by selective pulses (for the experiments performed here,COselective sinc pulses have been used), and the delays in the sequence have been adjusted to accommodate the much longer shaped pulses. The z-filters applied prior and after the indirect evolution have been omitted, as no significant effect on the spectra was observed. After acquisition, an nested inner loop is executed, which returns the execution of the sequence to the point directly after the hard pulse after cross polarization. During this time theCO magnetization is replenished via spin diffusion from the surrounding¹³Cnuclei. Then the sequence is repeated and the signals co added. After the magnetization of the neighboring nuclei is depleted, an outer loop, similar to the one in the normal Redor experiment, is performed, including a recycle delay for ¹H T1 relaxation and cross polarization. This repolarizes the whole spin system and a new set of inner loops can be performed. For this sequence, a rotor trigger was implemented directly after the z-filter to assure rotor synchronization of the sequence, irrespective of acquisition and RELOAD delays.

The enhancement gained for a band selectiveCO Redor RELOAD as depicted in Fig. 3.41 is shown in Fig. 3.42. In this figure it can be seen that the signal to noise ratio is significantly increased, without perturbation of the overall peak shape. The spectra have been scaled to the same level of noise.

The rise in signal to noise ratio with a increasing number of RELOAD cycles is shown in Fig. 3.43. Although the enhancement is not as pronounced as for the PDSD-RELOAD experiments shown in Figs. 3.31 and 3.32, enhancements of 1.4 in signal to noise could be achieved using four RELOAD cycles. The slope of this curve suggests even higher enhancements are possible, using a larger number of RELOAD cycles. As

Figure 3.43: Evaluation of the signal to noise enhancement over the number of RELOAD cycles for the spectrum shown in Fig. 3.42. The increase in signal to noise is significant, although it is less pronounced than for the RELOAD PDSD ex-periments shown in Fig. 3.31. Still an enhancement of 1.4 can be achieved for four RELOAD cycles.

The decrease in relative enhancement for the Redor-RELOAD, as compared to the PDSD-RELOAD, can be explained by the larger number of shaped pulses used in the former sequence. These could lead to a slight perturbation of the bath magnetization.

This perturbation is by far less pronounced as for the DCP-RELOAD experiment, and therefore significant gains in signal to noise can be achieved.

To investigate to which degree the bath magnetization is preserved, the experiment shown in Fig. 3.41 c) was employed. The sequence shown in b) was modified, so that the RELOAD cycle commences before the acquisition. Only after the last RELOAD loop, the bath magnetization is read out using a hard 90^◦pulse on¹³C.

Therefore the method of investigating the bath magnetization is a bit different to the one employed for the DCP experiment. Whereas, for the DCP, the accumulation of residual magnetization on the¹³C channel was observed, here the¹³C magnetiza-tion is dephased during the z-filter for all RELOAD cycles, save the last, where the magnetization for all¹³C spins is read out after all selective pulses have been applied for the last RELOAD loop. Insofar, this experiment can be regarded as even more stringent, as only the magnetization left after the last RELOAD cycle is recorded. The result of these measurements are shown in Fig. 3.44. Here, the residual magnetization of all¹³C resonances of the MLF spectrum is shown after the last RELOAD loop, as a function of total RELOAD loops. It is apparent that for most resonances, more that 50% of the initial magnetization is retained, even after five successive RELOAD cycles. This suggests that the Redor RELOAD sequence is only mildly perturbing to the bath magnetization, explaining the good gains in signal to noise ratio seen in Fig.

3.43.

These measurements suggest that Redor is a much better hetero-nuclear corre-lation sequence to be employed in conjunction with RELOAD, yielding significant enhancements, which allow a reduction in measurement time by about a factor of 2, with the current implementation.

3.6. APPLICATION OF RELOAD TO HETERO-NUCLEAR CORRELATION SPECTRA

Figure 3.44: Plot of the residual ¹³C magnetization after the last RELOAD cycle over the total number of RELOAD cycles, for all resonances of the MLF spectrum.

It is apparent, that even after five RELOAD loops, more than 50% of the initial magnetization is retained for most resonances. This implies a minor perturbation of the bath magnetization by the Redor RELOAD sequence. All spectra were recorded with at 600 MHz, 8 kHz spin rate, 295K and 4 scans. 50 Hz exponential line broadening was applied to all spectra.