199Hg NMR Standards

At this site we are posting discussions concerning 199Hg NMR standards. We welcome all comments about, and additions to, this page. We will try to update it as notes intended for posting are sent to us. Since you are visiting this site you are probably aware of the tragic death of Dr. Karen Wetterhahn, which was apparently caused by exposure to dimethylmercury. According to press accounts, exposure occured upon transfer of this liquid to an NMR tube while making an NMR reference standard. Apparently a few drops passed through the latex gloves she was wearing (see NOTE about gloves below). Since then, there have been several letters to Chemical and Engineering News containing suggestions and requests for the NMR community to adopt a new, less toxic 199Hg standard. On this page we will outline some of other methods of referencing 199Hg chemical shifts, as well as compounds that may serve as alternative references. Although the compounds presented here are easier to handle than dimethylmercury, some can still be hazardous to handle. In each case, investigators are recommended to review the material safety data sheets (MSDS) that accompany all chemicals before handling the material and to review the chemical permeability of the gloves to be worn. We will also post other investigators findings and suggestions here as we receive them.

Dimethylmercury as a reference

Many investigators have used external neat dimethylmercury as 1) a chemical shift reference, 2) a concentrated sample for the initial search for the 199Hg reference and 3) for measuring the inverse 90 pulse in two channel experiments. For instance, a small coaxial flame-sealed capillary inside a 5 mm NMR tube filled with D₂O is useful in a wide variety of experiments. As a reference material, the chemical shift of neat dimethylmercury is not prone to problems that arise with other compounds. For instance, the 199Hg chemical shift of many compounds exhibit a pronounced solvent, ionic strength, concentration, and pH dependence.

Dimethylmercury has several intrinsic properties that are well suited for other 199Hg experiments. A dense liquid at STP, it serves as one of the most concentrated forms of mercury nuclides aside from the elemental form. Why is this useful? Concentrated samples are essential when an investigator tries to observe a 199Hg resonance for the first time on a given instrument. Since the observation window and frequency offset need to be changed many times before the resonance for a given nuclide can be found, a highly concentrated sample makes each search quicker. The concentration of mercury in the neat dimethylmercury (13 M) is much higher than can be obtained with any mercury compound dissolved in a solvent. This feature allows spectra with good signal to noise ratio to be acquired with 1 scan, and an accurate 90° pulse can be determined quickly.

Finally, the proton spin systems in alkylmercury compounds like dimethylmercury exhibit substantial ³J ⁴J and ⁵J coupling to the 199Hg spin. This strong coupling (100 Hz) between the methyl protons and the mercury, allow for rapid and accurate measurement of the 199Hg (90)^-1 pulse in 2-dimensional experiments. Determination of this pulse width establishes the optimal parameters for coherence transfer NMR experiments.

Thus one compound serves many roles in 199Hg spectroscopic studies. However, given that dimethylmercury is such a toxic and volatile liquid, and that it is far more difficult and hazardous substance to handle than other mercury compounds, it is prudent to consider using other compounds in the above experiments. Most of the 199Hg chemical shifts in the literature to date are reported relative to dimethylmercury (1-7). In the future, we plan to make measurements relative to another standard and convert the values to the dimethylmercury scale, thus reporting 199Hg chemical shifts the same as in the past.

NOTE about gloves: Anyone considering handling dimethylmercury should consider the advice given by M. Blayney et. al. in a letter to Chemical and Engineering News ; May 12, 1997 p. 7). The authors recommend the following gloves: “A highly resistant laminate glove (SilverShield or 4H) should be worn under a pair of long-cuffed, unsupported neoprene, nitrile or similar heavy-duty gloves. Latex or PVC gloves have an important role in many laboratory activities, but they are not suitable for significant direct contact with aggressive or highly toxic chemicals.”

Alternative Hg compounds as a reference

Along with dimethylmercury, mercury perchlorate [Hg(ClO₄)₂] has been cited in the literature as a reference. At a concentration of 0.1 M in a 0.1 M perchloric acid solution the chemical shift is -2250, relative to dimethylmercury.(8) We have made up a Hg(ClO₄)₂ standard (in D₂O) according to these conditions and found the chemical shift to be within ±1 ppm of the quoted value, at T = 290-293 K. The resonance was observable in 1 scan.

This reference is a viable alternative to dimethylmercury. However, if 2-dimensional experiments are to be performed, determination of the inverse-90° pulse (90)^-1must be done. To do this accurately for a low abundance nucleus, it is necessary to have coupling between an abundant nucleus (1H) and the nucleus of interest(199Hg). Since mercury perchlorate is an ionic compound there is no coupling to the Hg and an accurate measurement of the inverse-90° pulse is not possible.

Because the NMR experiments on dilute samples of 199Hg-substituted proteins require 10-20 hours of spectrometer time, it is a good idea to measure the inverse-90° pulse. An accurate inverse-90° pulse increases the probability that a 2-D experiment will be successful. In our experience, we have seen this value fluctuate as a function of the tune and match of our 5 mm inverse detection probe with values ranging between 17-21 µs.

Alternative method for referencing chemical shifts.

In the July 14, 1997 issue of Chemical and Engineering News David Live and Robin Harris suggest that an indirect method for referencing chemical shift scales be used.(9) In their letters they describe the principle behind this method, which has been used in biomolecular NMR before. Additional helpful references can be found within those papers. This method would be an ideal way to reference chemical shifts, however it leaves several problems. In the situation of having a dilute sample (1-3 mM) and a nucleus with broad linewidths, it becomes important to know an accurate value for the 90° pulse for that nucleus, from an experimental time perspective.

Alternative organomercurials for inverse-90°determination.

Another potential candidate for a reference is p-chloromercuriphenyl-sulfonic acid. Although this compound is labeled “very toxic” (as any organo-mercurial is) it has the benefit of being a solid. Solutions of this compound can therefore be more safely and easily handled than dimethylmercury. The latter can permeate the skin. It is also volatile and can be inhaled. Of course, solutions of p-chloromercuriphenyl-sulfonic acid dissolved in some solvents (DMSO and methanol in particular) may be able to pass through the epidermis, so the appropriate gloves should be worn when making these solutions. A more comprehensive treatment of p-chloromercuriphenyl-sulfonic acid compound can be found in references to be posted later. We anticipate that the coupling between the phenyl 1H’s and the 199Hg will be large enough, so that measurement of the inverse-90° pulse will be possible. Our lab plans to evaluate the suitability of this compound as a 1- and 2-dimensional reference, and we will update this page when those experiments are completed.

In conclusion we would like to say that mercury-199 NMR can continue to be a useful spectroscopic tool in biochemical and biophysical research, when proper safety precautions are taken.

References

Please email comments or suggestions that you wish to add to this discussion to Thomas V. O’Halloran.

For more information about the O’Halloran group and their research see our homepage.

This page was created by Chris Singer on March 10, 1998 and maintained by David Zee.

Last Updated May 25, 2022