A dual-stage purge–trap protocol to separate species-specific mercury from marine biota for precise isotopic analysis†

The application of mercury (Hg) isotopes in marine food webs has greatly advanced our understanding of the sources and transformation of marine Hg. However, previous studies mainly focused on the total Hg (THg) isotope composition. While species-specific Hg isotope compositions are more informative, the separation of individual species remains a technological hurdle. Here, we optimized a dual-stage purge–trap protocol to separate inorganic Hg (IHg) and methylmercury (MeHg) from marine biota to facilitate high-precision isotopic analysis. First, the homogenized biological samples are dissolved in hydrochloric acid (HCl) and reduced by stannous chloride (SnCl₂) to convert IHg to Hg(0) vapor that is subsequently purged and trapped in the downstream trapping solution. Then, the sample residue is treated with potassium permanganate (KMnO₄) and potassium persulfate (K₂S₂O₈) to fully demethylate MeHg into IHg, which undergoes a second stage of SnCl₂ reduction and Hg(0) purging and trapping. The intermediate precision of this protocol was verified by monitoring Hg recoveries and isotopic compositions of both certified reference materials and natural samples. Our protocol provides the advantages of simultaneously separating IHg and MeHg from a single sample aliquot, featuring a rapid processing time of ∼3.5 h, a minimal procedural blank of ∼0.03 ng mL⁻¹, high recoveries (IHg: 96.2 ± 5.8% and MeHg: 96.8 ± 5.6%), and the capability to handle large volumes of samples.