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I am recording 15N-HSQC spectra of a protein in increasing concentrations (0.25–2.5 M) of guanidine hydrochloride. I would like to use the guanidine chemical shift (which appears in the HSQC) as an internal standard in both the direct and indirect (!) dimensions. My intuition is that the chemical shifts of guanidine should be independent of guanidine concentration. Does anyone know if this is really true?

Currently, I see a constant upfield shift in the indirect dimension, even for the guanidine peak itself, on the order of 0.3 ppm. The pH of the samples is constant, within error. The measurements are performed at the same temperature.

Thanks in advance!

asked Nov 17 '10 at 12:04

vadim's gravatar image


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With nothing better to say - maybe you can just try run a couple of 1D's of guanidine at different concentrations (for example - at the extremes of your set) with a proper chemical shift (like DSS 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) or TSP 3-trimethylsilylpropionate-d6) added and just see what the shift will be?

Gu's H will experience difference in the interaction with water in addition to change of field because of changing magnetic susceptibility (the more guanidinium is added), while DSS will only experience the latter, so Guanidinium will be a poor standard of chemical shift even at constant pH and temperature.


answered Nov 19 '10 at 10:09

Evgeny%20Fadeev's gravatar image

Evgeny Fadeev

updated Nov 19 '10 at 12:06

Samples contain DSS as an internal reference. I know that the chemical shift of DSS is not sensitive to pH or temperature. However, since the Gua resonance is changing (slightly) relative to the DSS, my question really is about whether the DSS resonance is sensitive to Gua. - vadim (Nov 19 '10 at 11:20)

I am also curious whether the chemical shift of Gua is sensitive its own concentration. If not, I can use it as a convenient internal reference. For instance, is the chemical shift of methanol (i.e.) different at different methanol concentrations (v/v) ? - vadim (Nov 19 '10 at 11:22)

well, imo the best bet is to use an inert compound like DSS as a reference for the 1H scale and gyromagnetic ratios of 1H and 15N to re-reference the Hz value of 0ppm of 15N scale. Probably guanidinum changes magnetic susceptibility of the sample and the resonance frequencies shift as a result. - Evgeny Fadeev (Nov 19 '10 at 11:57)

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