Cloud point extraction preconcentration prior to high-performance liquid chromatography coupled with cold vapor generation atomic fluorescence spectrometry for speciation analysis of mercury in fish samples

A cloud point extraction methodology was developed for simultaneous preconcentration of Hg(II), methylmercury (MeHg), ethylmercury (EtHg), and phenylmercury (PhHg) prior to reversed-phase high-performance liquid chromatography (HPLC) on-line coupled with cold vapor atomic fluorescence spectrometry for speciation analysis of mercury in fish. The four mercury species were taken into complexes with ammonium pyrrolidine dithiocarbamate (APDC) in aqueous nonionic surfactant Triton X-114 medium and concentrated in the surfactant-rich phase by bringing the solution to the temperature of 40 degrees C. Baseline separation of the enriched complexes was achieved on an RP-C-18 column with a mixture of methanol, acetonitrile, and water (65:15:20, v/v) containing 200 mmol L-1 HAc (pH 3.5) as the mobile phase. An on-line postcolumn oxidation of the effluent from HPLC, in the presence of K2S2O8 in HCl, was applied in the system followed by an optimal cold vapor generation of mercury species. The variables affecting the complexation and extraction steps were examined. The preconcentration of 10 mL of solution with 0.08% w/v Triton X-114 and 0.04% w/v APDC at pH 3.5 gave enrichment factors of 29, 43, 80, and 98 for MeHg, EtHg, PhHg, and Hg(II), respectively. Low detection limits (S/N = 3) were obtained, ranging from 2 to 9 ng L-1 (as Hg) for all species. The developed method was successfully applied to the speciation of mercury in real fish samples.