I recorded T1 and T2 relaxation data at two different fields (600 and 750 Mhz) for model-free analysis. A prerequisite is to have absolutely the same experimental conditions at any time during the measurements. This is also true for the temperature at different machines.
We use d4-methanol to calibrate different spectra to the same temperature, which seems to work pretty well overall. However, I expected to get exactly the same TROSY of the same sample at every spectrometer. But that's not always the case: Some signals are diverging from the expected positions, i.e. most of the signals have identical positions at 600 and 750Mhz, but a small number of them don't.
I can imagine this is due to changes in the sample (hydrolysis (?), aggregation side effects, etc), but is there maybe an alternative explanation?
Are there field-dependent effects that could explain these shifts of the signals, or should every TROSY be 100% identical to the other, regardless of the external field?
Are these two samples slit from the same stock or prepared separately? If prepared separately, my first guess would be that the pH is slightly different between the two samples.
Aggregation can be checked by a DOSY experiment to measure the hydrodynamic radius.
As far as field effects on the spectra, changing the field could shift chemical exchange of some of the peaks from the slow to intermediate regime at higher fields. If the population is highly skewed towards one conformation, the minor peak may not be visible in the slow exchange regime. The peak would then appear to change position at higher field. See Applied Geochemistry Volume 22, Issue 8, August 2007, Pages 1612–1623 for a simple example, a full explanation can be found in J. Am. Chem. Soc., 122 (2000), pp. 2867–2877
Hi Jeffrey,
thank you for your answer,
In this particular case, the sample in the same one, so different pH values shouldn't be an issue. Chemical exchange and the depency on field strength is a good point, I wasn't thinking of that.
Thanks again
Martin
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