In NMR spectra solvent peaks often obscure the peaks of interest. Using deuterated solvents is one way to avoid this problem, but solubility or the need to observe exchangeable protons such as hydroxyls and amides sometimes requires using a protonated solvent. This is often the case when trying to use physiological conditions, as in biomolecular NMR or metabolomics. To detect the solute in aqueous solutions the water peak must be suppressed and there are several different ways this can be achieved.
To demonstrate some solvent suppression techniques I used a sample containing 2.0 mM sucrose in 90% H20/10% D2O with 0.5 mM DSS. The water peak appears at 4.7 ppm, the sucrose peaks between 5.5 and 3.0 ppm, and the DSS peaks between 3.0 and 0.0 ppm.
In the figure below the four spectra have been scaled so that the DSS peak at 0.0 ppm has the same intensity. The bottom spectrum (blue) is a 1D 1H spectrum without solvent suppression. The water peak dominates the spectrum. The sucrose peaks can still be observed between 4.5 and 3.0 ppm but they are hard to interpret and cannot be integrated without extensive baseline correction. The second spectrum (red) was recorded with presaturation of the water peak. While the water peak is still present, the baseline is much flatter allowing integration of the sucrose peaks, and the anomeric proton at 5.40 ppm can be much more easily observed. The third spectrum (green) used excitation sculpting to suppress the water. This spectrum is very similar to the presaturation spectrum with a slightly wider water peak but better signal-to-noise. The upper spectrum (maroon) was recorded with the jump-return method and did not work well for this sample.
Presaturation1 works by applying continuous low power irradiation during the relaxation delay to scramble the water magnetisation so that it is no longer coherent. This greatly reduces the signal it produces. The longer the irradiation period, the narrower the signal suppression region. The WET experiment2 is a modified presaturation sequence in which multiple sites are irradiated during the relaxation delay. This allows multiple solvent signals to be suppressed.
Excitation sculpting is a form of WATERGATE.3 WATERGATE uses a spin echo composed of a selective composite pulse that inverts all magnetisation, except the water, flanked by a pair of gradients to recover the inverted magnetisation. Excitation sculpting4 improves on WATERGATE by repeating the spin echo to give a flatter baseline and improved phase. It is possible to replace the selective composite pulse in the spin echo with a water selective 180o pulse followed by a non-selective 180o pulse. This method is less sensitive to miscalibration and was the method used here.
Presaturation and excitation sculpting both have their drawbacks. Since presaturation destroys the water magnetisation it also reduces the signals of protons that exchange with the water. Depending on the exchange rate and length of presaturation, much of the exchangeable signal can be lost. In the excitation sculpting method the gradient pairs act a little like a DOSY sequence so that, for small molecules in particular, the signal is attenuated due to diffusion. The jump-return sequence5 does not suffer from these drawbacks. It uses a pair of 90o pulses 180o out of phase so that the on-resonance water signal is not excited. The spectrum obtained is effectively scaled by a sine wave so that the center is zero, one side of the spectrum is positive, and the other negative. This is particularly useful for samples with exchangeable peaks far from the water, such as nucleic acids.
All of these techniques can be used for suppressing solvents other than water, for example methanol, and can be integrated into 2D and 3D experiments. The Skaggs NMR Facility has presaturation and excitation sculpting versions of most 2D standard parameter sets as well as 1D 1H versions of presaturation, excitation sculpting and jump-return.
References
1. Selective saturation of carbon-13 lines in carbon-13 Fourier transform NMR experiments
Jacob Schaefer
J Magn Reson, 1972;6(4):670-671
2. WET, a T1- and B1-Insensitive Water-Suppression Method for in Vivo Localized 1H NMR Spectroscopy,
R.J. Ogg, R.B. Kingsley, J.S. Taylor,
J Magn Reson B, 1994;104(1):1-10
3. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions.
Piotto M, Saudek V, Sklenár V.
J Biomol NMR, 1992 Nov;2(6):661-5
4. Water Suppression That Works. Excitation Sculpting Using Arbitrary Wave-Forms and Pulsed-Field Gradients,
T.L. Hwang, A.J. Shaka
J Magn Reson A, 1995;112(2):275-279
5. Exchangeable proton NMR without base-line distorsion, using new strong-pulse sequences
Pierre Plateau and Maurice Gueron
J Am Chem Soc 1982;104(25):7310-7311
No comments:
Post a Comment