Improved RP-HPLC separation of Hg2+ and CH3Hg+ using a mixture of thiol-based mobile phase additives

Hg²⁺ and CH₃Hg⁺ are frequently encountered in the environment either as free ions or complexed with organic matter, such as humic acids. The majority of the reported HPLC-based separations of environmental mercury species, however, separate Hg²⁺ from CH₃Hg⁺ in which the former species elutes close to the void volume. To detect mercury-species in environmental waters that may have so far escaped detection, a separation method is needed that sufficiently retains both Hg²⁺ and CH₃Hg⁺. One way to develop such a method is to increase the retention of Hg²⁺ and CH₃Hg⁺ using existing HPLC separations. We here report on the improvement of a previously reported RP-HPLC-based separation of Hg²⁺ and CH₃Hg⁺ that employed a 100 % aqueous mobile phase [10 mM L-cysteine (Cys) in 50 mM phosphate buffer (pH 7.5)]. To increase the retention of Hg²⁺, Cys was replaced by the comparatively more hydrophobic N-acetylcysteine (N-Cys). To achieve a compromise between an increased retention of Hg²⁺ and its baseline separation from CH₃Hg⁺ in the shortest possible analysis time, the retention behavior of both mercurials was investigated on two RP-HPLC columns with mobile phases that contained mixtures of Cys and N-Cys in which the overall thiol concentration was maintained at 10 mM. An optimal separation of both mercurials could be achieved in ∼540 s using a Gemini C₁₈ HPLC column (150 × 4.6 mm I.D.) and a mobile phase comprised of 7.5 mM N-Cys and 2.5 Cys in 50 mM phosphate buffer (pH 7.4). Coupling the developed HPLC separation with an inductively coupled plasma mass spectrometer should allow one to detect mercury species other than Hg²⁺ and CH₃Hg⁺ in environmental waters. The detection of such species is critical to better understand the mobilization of mercury species from natural and anthropogenic pollution sources.