Improved Solvent Suppression with Composite Pulses

A hard rf pulse delivers an rf field to the NMR sample inside of the coil. The rf field is not perfectly homogeneous nor does it end abruptly at the edge of the coil. When a long, selective, low power presaturation pulse is given to suppress a water signal, the water in the coil may be fully saturated whereas water outside of the coil will not be. Furthermore, unsaturated water outside of the coil may be outside of the carefully shimmed region of the magnet and give rise to a broadened residual signal when presaturation is used prior to a hard 90° excitation pulse. One long known* way to avoid this residual broad signal is to use a composite 90° pulse after presaturation.  One of the simplest such pulses is a (90°_x - 90°_y - 90°_-x - 90°_-y) composite pulse which is designed to excite sample inside of the coil but not outside of the coil. As a result, when it is used after a water presaturation pulse, it will not excite the broad signal from the unsaturated water outside of the coil and it will provide a spectrum with better presaturation performance. The figure below shows a small portion of the ¹H spectrum of a plant extract in H₂O/D₂O.

The water signal is highlighted in pink. The left panel shows a conventional spectrum acquired with a 90° pulse. The center panel is a spectrum of the same sample where a two second low power presaturation pulse preceded the 90° hard pulse (Bruker pulse program = zgpr). One can see that most of the water is suppressed from the presaturation pulse however, a broad water signal remains from the water outside of the coil. The spectrum in the right-hand panel is the same as that in the center except that the 90° pulse was replaced with a composite 90° pulse (Bruker pulse program = zgcppr). Clearly, the broad signal from the unsaturated water outside of the coil is essentially gone providing a spectrum with much better water suppression.

*A. Bax.  J. Magn. Res. 65, 142 (1985).

"Absolute" Water Suppression

Collecting ¹H NMR spectra of aqueous samples is complicated by the presence of the enormous, broad water signal which is often many orders of magnitude more intense than the signals from the solute of interest. The water signal can be suppressed by presaturation or multiple pulse techniques employing gradients (such as WATERGATE). These techniques are compared here and do a very good job, but neither is able to completely suppress the water signal in very dilute challenging samples. Recently, a technique introduced by Buuan Lam and Andre Simpson* which uses both presaturation and W5-WATERGATE has been used to suppress the water signal. In this case, the presaturation consists of many low power shaped 180° pulses. The combination of these two techniques provides incredible water suppression. The authors are able to measure the ¹H NMR spectra of dissolved organic matter in natural waters without any preconcentration of the samples. Their spectra are completely free of the water signal! At the 2011 ENC the same group presented a poster** highlighting the water-free ¹H NMR spectrum of the dissolved organic matter in melted glacial ice. The water suppression is so remarkable that this technique should be called "The Simpson Sledgehammer". The figure below illustrates further examples of the implementation of this suppression method. The bottom trace shows the 32 scan ¹H NMR spectrum of a dilute unlabelled protein (which includes the NH resonances, albeit attenuated due to chemical exchange). The middle trace shows the 32 scan ¹H NMR spectrum of supernatant human saliva. The upper trace shows the ¹H NMR spectrum of rain water collected from an asphalt tile roof. The spectrum was collected overnight and shows the presence of long chain hydrocarbons from the asphalt as well as formaldehyde. In all cases the water signal is completely absent in the spectrum.

* Buuan Lam and Andre J. Simpson. Direct ¹H NMR Spectroscopy of Dissolved Organic Matter in Natural Waters. The Analyst 113 263 (2008).

** Brent Pautler, Andre Simpson, Li-Hong Tseng, Manfred Spraul, Ashley Dubnick, Martin Sharp and Myrna Simpson. Trace Level Analysis of Dissolved Organic Matter in Glacial Ice Using SPR-W5-WATERGATE. POSTER 264, ENC (2011).

Watergate vs Presaturation

Biochemists and protein chemists are often interested in observing the NH protons in their samples. Since the NH protons usually undergo slow chemical exchange with water, it is desirable to run the samples in H₂O rather than D₂O so the NH protons will not exchange with the deuterium in the solvent which would make them invisible in the ¹H NMR spectrum. In practice, a mixture of 10% D₂O and 90% H₂O is used as a solvent so that a deuterium lock can be established and used while running the spectrum. The very high concentration of water compared to the very low concentration of solute necessitates the use of solvent suppression methods.

Both presaturation and WATERGATE are efficient techniques used to suppress strong water signals from proton NMR spectra, however, there are differences between the two methods of which the user must be aware. Presaturation employs a selective, long, low power pulse to saturate the water resonance. This pulse is usually several seconds in duration during which exchange can occur between the unsaturated NH protons and the saturated water protons. If this exchange occurs, the NH protons become partially saturated to an extent related to the rate of chemical exchange between the NH and the water. The intensity of the NH protons in the spectrum is non-quantitative. WATERGATE, on the other hand, uses a pair of gradients surrounding a composite pulse which in effect inverts all but the water signal. The duration of the composite pulse is about 4 orders of magnitude shorter than a presaturation pulse, so exchange between the NH protons and the solvent occurs to a much lesser extent during the WATERGATE sequence compared to presaturation. As a result, the NH region is much less attenuated and more quantitative in a spectrum collected using WATERGATE compared to a similar spectrum run with presaturation. This is illustrated in the figure below which shows the NH region of the ¹H spectrum of a small disaccharide substituted peptide.One can see that some of the NH's are greatly attenuated in the spectrum acquired with presaturation compared to the spectrum run using WATERGATE. The more attenuated the signal, the faster the chemical exchange for that particular NH with water.

The WATERGATE suppression sequence is very similar to the gradient spin echo sequences used to measure diffusion constants and DOSY spectra. As a result, when WATERGATE suppression is used, one expects diffusion losses for small molecules (which diffuse quickly) compared to large molecules (which diffuse slowly). This effect is demonstrated in the figure below which shows the NH/aromatic region of the ¹H spectrum of a partially degraded ¹⁵N labelled 10 kDa protein.Here, one can see the same exchange losses pointed out in the previous figure for the presaturation spectrum compared to the WATERGATE spectrum. In addition, one can see that the intensity of a very sharp peak marked in yellow (likely due to a CH proton from free histidine) is less intense in the WATERGATE spectrum compared to the presaturation spectrum. This loss is due to the fast diffusion of the small free amino acid compared to the very large protein. In conclusion, one must be aware of the differences between the two solvent suppression methods if quantitative results are being sought.

I would like to thank Roger Tam from Robert Ben's Laboratory and Allison Sherratt from Natalie Goto's Laboratory for kindly providing the samples of the peptide and protein, respectively.

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.

WATERGATE

WATER suppression by GrAdient Tailored Excitation (WATERGATE) is a clever technique used to suppress the water signal in an aqueous sample. It is widely used in many complicated pulse sequences. Unlike presaturation which irradiates the water resonance with a long low power pulse, this method is based on the gradient spin echo technique used also to measure diffusion constants and DOSY spectra. The pulse sequence is shown here:The transmitter frequency is set on the water resonance. A non-selective hard 90° pulse is applied followed by a 1 -2 msec gradient pulse. The gradient pulse dephases all of the resonances. A composite pulse (consisting of 6 hard pulses seperated by a delay, τ) is then applied which acts as a 180° pulse for everything except peaks on resonance (i.e. water) and any peaks at frequencies n/τ away from the transmitter, where n is an integer. τ is chosen such that 1/τ lies outside of the spectral width (typically several hundred µsec). The second gradient pulse (equal in magnitude, duration and sign, to the first) further dephases the water resonance at the center of the spectrum which was unaffected by the composite pulse but rephases everything else which was inverted by the composite pulse. The gradients and composite pulse act as a gradient spin echo for all but the water. The FID is then collected with the water resonance suppressed by the two dephasing gradients. An example of the application of WATERGATE is shown in the figure below. The top trace shows a standard 500 MHz ¹H NMR spectrum of phenylalanine in H₂O / D₂O scaled to the water peak. The resonances of the phenylalanine are not visible on this scale. The middle trace is the same spectrum as the top trace with the phenylalanine resonances on scale. The huge water resonance is truncated. The bottom trace shows the WATERGATE spectrum. The water signal is greatly suppressed.