ROESY mixing time

The ROESY used to be a bit difficult to set up because the low power spin-lock RF had to come from a different source than the hard pulse RF. Now rapid solid-state power switching is so routine that all H RF comes from the same source, with no variation of phase or frequency. ROESY tends to replace the NOESY experiment for NOE measurements, especially for small molecules where T2 is relatively long. Because the NOE builds up about twice as fast in the x -y plane as it does on the z axis, ROESY mixing times are set to about half of what would be the NOESY mixing time. There is one additional parameter to set up the power level ( Z i field strength ) of the spin lock pulse. This is typically... 

The easiest way to reduce the amplitude of TOCSY cross peaks in the ROESY spectra is to record a spectrum with minimal spin-lock power [23]. The other possibility is to modulate the frequency of the spin-lock field [25]. However, the most convenient way is to apply a series of 180° pulses instead of a single continuous-wave pulse during the mixing time, as is done in the T-ROESY experiment. Figure 4(D) shows the T-ROESY spectrum of cyclo(Pro-Gly) recorded with = 300 ms. Although the... 

In contrast to the 1D experiment, where steady-state NOEs may be obtained, only the less intense transient NOE.s are measured in the NOESY experiment. ROEs can only be obtained as transient effects in both the ID and the 2D experiment. Furthermore the intensities of the NOESY and ROESY cross peaks depend upon the molecular size as well as the length of the mixing period. In the case of large molecules, e.g. polypeptides, rather short mixing times are usually chosen to avoid spin diffusion. 

MLEV17 sequence being one of the most used sequences [23]) in such a way as to continuously refocus the chemical shift evolution of the various signals in the xy plane. Analogously to ROESY experiments, the magnetization during the spin-lock (mixing) time disappears with T p (i.e. essentially Tj, see Section 3.4). It follows that coherence transfer in the xy plane, which is built up with a sin(7T J/jt) function, also decreases with time constant p p — p[p + p p)/2 ... 

The same considerations that are relevant for the selection of mixing times and relaxation delay times for NOESY experiments also apply to ROESY [i.e., suggested RT s are about 2ri for small to intermediate-sized molecules (MW 400-750) and should generally be in the 0.9-1.8-s range (Section 7-lOa)]. Spin-lock mixing times are in the 100-500-ms range—closer to 100 ms for larger molecules and 400-500 ms for smaller molecules. 

Figure 8.45. The 2D ROESY sequence. The mixing time, tm. is defined by the duration of the low-power spin-lock pulse.

To establish interpulse delays for two-dimensional NMR experiments, it is frequently convenient to run a very quick proton Tj relaxation measurement. Given the sensitivity of modem spectrometers, this can usually be done with only a single or a few transients for each of the r values in the series, and typically requires 10 min or less. By visual inspection, the Tj relaxation time can be estimated from the r value at which response intensity is zero. A knowledge of the Tj relaxation time is also useful for establishing mixing times for NOESY and ROESY experiments. 

Fig. 8.17 ROESY spectrum of the aliphatic region of strychnine (2) recorded with a 250 ms mixing time. The correlations observed and the assignment information which can be derived from them is the same as for the NOESY experiment presented in Fig. 8.15.

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