Chemical Shift Standards for ¹⁹⁹Hg NMR Spectroscopy, Twenty-Five Years Later

Abstract

While mercury-199 NMR is a well-established tool for elucidating details of coordination chemistry in biochemical and inorganic complexes, historically the technique has been associated with the use of the extremely toxic chemical, dimethylmercury, as a reference standard. In the twenty-five years since an accidental exposure to dimethylmercury led to the tragic death of Dr. Karen Wetterhahn, the community has learned a great deal about the insidious neurotoxicity of this compound as well as more appropriate ways to avoid exposure. Here, we track the general shift towards the use of alternative mercury reference standards, and away from dimethylmercury.

INTRODUCTION

Twenty-five years ago this June, the inorganic chemistry community suffered the loss of an extraordinary scholar, teacher, and mentor, Dr. Karen Wetterhahn, who died many months after an accidental exposure to dimethylmercury. A few drops of this lipid-soluble organometallic reagent fell upon, and passed through, the latex glove she wore while preparing it for use as a standard for a series of ¹⁹⁹Hg NMR experiments. The courage Dr. Wetterhahn showed in insisting that details of her accident be shared after her death, as well as the science underpinning the slow manifestation of dimethylmercury toxicity, has been described in a number of first-hand accounts from colleagues and health safety professionals (1–5). Since it is hard to imagine how such a small exposure on the skin could have such profound effects, it is helpful to review the analytical data. The initial blood test, performed 162 days after Dr. Wetterhahn’s accident, showed her blood mercury level to be 4,000 μg/L, well over the normal range of 1–10 μg/L (3, 6). Elemental analysis of hair samples as a function of growth rate indicated a single exposure within the time frame annotated in Dr. Wetterhahn’s notebook for preparation of the NMR sample (3). Considering that 0.1 mL of Me₂Hg fully diluted into the 5 L of blood in an adult corresponds to ca. 60,000 μg Hg/L. Given the 2–3 month half-life for Me₂Hg clearance, Nierenberg and coworkers concluded that absorption of 0.1–0.4 mL of Me₂Hg in a single exposure was sufficient to account for the blood data and Dr. Wetterhahn’s symptoms (3, 6). While the materials safety data sheets at that time did not specify types of gloves that afford appropriate protection, Blayney has since shown that almost all types of laboratory gloves are permeable to dimethylmercury and that a combination of Silver Shield and neoprene gloves is now specified for optimal protection (7). In this Viewpoint we update and expand upon a discussion of alternative chemical shift standards that was first posted as a website at the time of Dr. Wetterhahn’s death (8). After addressing why dimethylmercury was employed in the initial set up of ¹⁹⁹Hg NMR experiments and as a chemical shift standard, we outline the utility of alternative reference compounds and provide recent examples from the literature. Given what the toxicology and chemical health and safety communities have learned from Dr. Wetterhahn’s case about the extraordinary risks of handling dimethylmercury, and given the clear utility of alternative mercury-containing compounds as standards, we underscore and amplify a simple message: dimethylmercury need not be used in most ¹⁹⁹Hg NMR experiments.

DIMETHYLMERCURY AS A CHEMICAL SHIFT REFERENCE

Multinuclear NMR experiments are a cornerstone for understanding the solution and solid-state chemistry of over 90 elements in the periodic table (9). Our understanding of Hg(II) chemistry in particular has benefited from studies employing the ¹⁹⁹Hg isotope, which has a nuclear spin of 1/2 (I = 1/2), a natural abundance of just under 17%, and is readily available in a highly enriched state in a number of compounds. The chemical shifts of Hg(II) compounds are extremely sensitive to changes in the coordination environment (i.e., ligand type and binding geometry) which opens many doors in terms of the discovery of new mercury chemistry (10–23).

Dimethylmercury has several intrinsic properties that make it well-suited for a variety of ¹⁹⁹Hg NMR experiments. These properties led to the use of neat solutions of dimethylmercury in multinuclear NMR experiments as 1) a chemical shift reference standard, 2) a concentrated sample for the initial search for the ¹⁹⁹Hg reference signal, and 3) for determining the appropriate 90° pulse width in one dimensional experiments and the inverse-90° pulse in two channel experiments. Advances in multichannel NMR probe design provides alternative approaches in the latter case; however, it will be some time before instruments currently in use are replaced.

As a reference material, neat dimethylmercury is attractive as it is not prone to problems that arise with solutions of more labile mercury compounds. For instance, the ¹⁹⁹Hg chemical shift of many compounds exhibit a pronounced dependence on solvent, temperature, ionic strength, concentration, and pH (24–25).

As a dense liquid at ambient laboratory conditions, dimethylmercury serves as one of the most concentrated forms of mercury nuclides aside from elemental mercury. 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. Like many NMR-active isotopes of third-row elements, the range of ¹⁹⁹Hg chemical shifts can span thousands of ppm. Since the observation window and frequency offset may need to be changed many times before the resonance for a given nuclide can be observed for the first time on an instrument, a highly concentrated sample makes each search quicker. The concentration of mercury in 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 traditional Hg-coordination compounds, organomercurials (like dimethylmercury), and Hg-protein and -nucleic acid complexes exhibit substantial ²J, ³J, ⁴J, and even ⁵J coupling to the ¹⁹⁹Hg spin. These features make ¹⁹⁹Hg NMR an exceptionally powerful tool for understanding the solution and solid-state chemistry of mercury-containing compounds. Dimethylmercury has strong ²J coupling (100 Hz) between the methyl protons and the mercury nucleus, allowing rapid and accurate measurement of the ¹⁹⁹Hg inverse 90° pulse in two-dimensional experiments. Determination of this pulse width establishes the optimal parameters for coherence transfer NMR experiments.

Thus, with dimethylmercury 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 most other mercury-containing compounds, it is prudent to consider using other compounds in the above experiments.

ALTERNATIVE MERCURIAL COMPOUNDS FOR CHEMICAL SHIFT REFERENCING

While most of the ¹⁹⁹Hg chemical shifts in the literature are reported relative to dimethylmercury (16–18, 26–29), measurements can be made using alternative, safer standards that can be easily converted to the dimethylmercury scale. We examined the literature to evaluate how the community has referenced ¹⁹⁹Hg NMR data (see Supporting Information for full details on the literature search). Since 1997, neat dimethylmercury remains the most frequently used external reference standard (as recently as 2021, see Figure 1). Nevertheless, mercury perchlorate has been successfully used in the literature as a chemical shift reference (15, 25, 30–33), even though its chemical shift is concentration dependent (twelve instances, see Figure 1). At a concentration of 0.1 M Hg(ClO₄)₂ in a 0.1 M perchloric acid solution the chemical shift is −2250 ppm relative to dimethylmercury (25, 30). Our lab has made Hg(ClO₄)₂ standards 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 one scan.

Figure 1.

Publications from 1997 to present that feature ¹⁹⁹Hg NMR measurements, categorized by the mercury compounds used as reference standards (see Supporting Information for the methodology used for the literature search). Not specified = the authors did not mention what internal or external reference standards were used. Calculated = the ¹⁹⁹Hg chemical shifts were referred to Me₂Hg (δ = 0.0) with a calculation procedure using Ξ(¹⁹⁹Hg) = 17.910.822 % (20, 42–44). Total publications = 106.

Aqueous solutions of mercuric chloride have also been used as chemical shift standards for ¹⁹⁹Hg NMR (used in 16 publications since 1997, see Figure 1). Saturated solutions of HgCl₂ in either D₂O (−1550 ppm versus Me₂Hg) or dimethylsulfoxide-d₆ (−1501.6 ppm versus Me₂Hg) have been used (34–41).

In 2001, IUPAC endorsed the unified chemical shift scale, where chemical shifts of all nuclei are reported relative to the proton resonance of tetramethylsilane diluted in CDCl₃ (42–43). That chemical shift (versus Me₄Si) can then be converted to a secondary reference of the nuclei of interest using published Ξ values. Three publications adopted this method to reference the Hg chemical shift of their compounds (see Figure 1).

Unfortunately, concentrated solutions of HgCl₂ and Hg(ClO₄)₂ are not useful alternatives to neat dimethylmercury in heteronuclear correlation experiments, which require determination of an inverse-90° pulse. To do this accurately for a low abundance nucleus, it is necessary to have reasonable coupling between the abundant nucleus (i.e., ¹H) and the nucleus of interest (¹⁹⁹Hg). The alternative standards discussed above are ionic compounds that lack non-exchanging protons that couple with the ¹⁹⁹Hg nuclei, and an accurate measurement of the inverse-90° pulse is not possible. Because the NMR experiments on dilute samples of ¹⁹⁹Hg compounds such as Hg-proteins and complexes can require a dozen hours or more of spectrometer time, a well-established value for the inverse-90° pulse increases the probability that a 2-D experiment will be successful. In our experience, this value can fluctuate (as a function of the tune and match for our probe) between 17–21 μs (17–18, 29). We have no experience with alternatives to dimethylmercury for inverse-90° determinations; however, solutions of p-chloromercuriphenyl-sulfonic acid or methylmercury chloride should be considered. Although these compounds are labeled “very toxic” (as any organo-mercurial is), they can be more safely and easily handled than neat dimethylmercury.

It is somewhat surprising that only a few of the 3,000 papers published on the preparation of mercury coordination compounds and organomercurials since 1997 present ¹⁹⁹Hg NMR data (ca. 3 % of publications). This can be partly attributed to the difficulty of obtaining a ¹⁹⁹Hg NMR spectrum of some kinetically labile and/or low coordinate Hg compounds where contributions of chemical exchange and/or chemical shift anisotropy can broaden the ¹⁹⁹Hg NMR signal (45–49).

Regardless of the challenges that might remain in establishing a safer and universally accepted alternative ¹⁹⁹Hg NMR reference standard to dimethylmercury, the publications identified in Figure 1 clearly show its use is diminishing. From this, it seems clear that by choosing to share her story twenty-five years ago, the message Dr. Wetterhahn wished to convey to others in the scientific community has been heard and heeded.

CONCLUSION

In conclusion, applications of mercury-199 NMR continue to expand our understanding of inorganic chemistry, across solution chemistry, bioinorganic studies, solid-state and materials chemistry, and in biochemical and biophysical research. While it is desirable to continue reporting chemical shifts relative to dimethylmercury, we encourage the community use either the IUPAC unified scale approach or, when needed, a different experimental standard compound. Furthermore, we encourage researchers to explicitly annotate their referencing method in the experimental section of manuscripts. We anticipate that this Viewpoint will help the experimentalist explore safer alternatives to dimethylmercury.