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Thursday, February 11, 2010

Presaturation

One of the simplest and widely used ways to eliminate a strong water signal is to use presaturation. In this technique, the transmitter is set to the water resonance. a very long (seconds) low power (mW) pulse is given. The excitation profile of this pulse is very narrow due to its length and it saturates the water resonance at the transmitter frequency. A non-selective hard 90° pulse (with a wide excitation profile) is then given to place all remaining spins in the transverse plane for detection. An example of this is shown in the figure below. The top trace is a standard 500 MHz 1H NMR spectrum of phenylalanine in 90% H2O / 10% D2O. The resonance due to the water is huge and off-scale in the figure. The bottom trace is the same sample run with presatutation.

4 comments:

Anonymous said...

Hi Glenn. What is ment by "saturates the water resonance". Does the long very selective pulse on the water-resonance not also flip the water proteins into the transverse plane?

Glenn Facey said...

Anonymous,
Perhaps these posts will help explain the concept of saturation:

https://u-of-o-nmr-facility.blogspot.com/2018/03/appropriate-choice-of-presaturation-time.html

https://u-of-o-nmr-facility.blogspot.com/2018/02/how-much-presaturation-power-is-needed.html

I'm not sure what you mean by "water proteins".

Glenn

Anonymous said...

Dear Glenn. Thank you for your blog, I would require help from an expert to understand a strange effect of presaturation on the integration of certain signals.
I'm measuring thiol/disulfide mixtures (glutathione and mercaptoethanol) in 90%H2O/10%D2O phosphate buffer (100 mM, pH = 7.0) at 600 MHz. Without presaturation, all is fine (except low signal to noise). With presaturation, the CH2-group adjacent to the SH is ca. 20% smaller than it should be in glutathione, ca. 10% for mercaptoethanol. This effect is only observed for the thiols, with the disulfides all is fine.
What could be the reason for this? I was thinking about an NOE, but for these small molecules it should be positive, not negative.
I really would like to understand what's going on, but I couldn't find an answer.
Thanks if you would have insights into this.
BR, Silvan

Glenn Facey said...

Silvan,
If the methylene signals are not close to the water resonance being presaturated and they do not exchange with water (I can't imagine why they would) then I have no explanation as to why the signals would be underrepresented in the spectra employing presaturation. Are the intensities altered with varying presaturation power or duration? Have you tried using other water suppression methods like WATERGATE?

Glenn