Abstract Hollow-cathode (HC) radiofrequency glow-dis- charge (rf-GD) optical-emission spectrometry (OES) has been used as detector for the determination of inorganic mercury by cold-vapour (CV) generation in a flow-injec- tion (FI) system. Both NaBH 4 and SnCl 2 were evaluated as reducing reagents for production of mercury CV. The conditions governing the discharge (pressure, He flow rate, and delivered power) and Hg CV generation (NaBH 4 or SnCl 2 concentration and reagent flow rate) were opti- mized using both reducing agents. The analytical perfor- mance characteristics of FI–CV–rf-GD–OES for mercury detection were evaluated at the 253.6 nm emission mercury line. Detection limits (DL) of 0.2 ng mL –1 using SnCl 2 and 1.8 ng mL –1 using NaBH 4 were obtained (100 μL sample injections were used). When the optimized experimental conditions using SnCl 2 had been determined, the analytical potential of this CV–rf-GD–OES method was investigated as on-line de- tector for high-performance liquid chromatographic (HPLC) speciation of mercury (Hg(II) and methylmercury). The HPLC–CV–rf-GD–OES detection limits for 100 μL sam- ple injections were found to be 1.2 and 1.8 ng mL –1 (as mercury) of inorganic mercury and methylmercury, re- spectively. The reproducibility observed was below ±8% for both species. Finally, the HPLC–CV–rf-GD–OES sys- tem developed was successfully applied to the determina- tion of methylmercury (speciation) in two certified refer- ence materials, Dorm-2 and Dolt-2. Introduction The Environmental Protection Agency (EPA) regards mercury as a toxic element with accumulative and persis- tent character in the environment and biota. It is also well known that such toxicity depends upon its chemical form in a given biological sample [1]. Methylmercury is by far the most toxic mercury species because it can cross bio- logical membranes more easily than the corresponding in- organic forms. For this reason information about mercury speciation is increasingly needed nowadays. It is undeniable that the most powerful instrumental approach to speciation analysis is coupling a chromato- graphic technique with an atomic detector (i.e. hybrid or hyphenated techniques). Gas chromatography (GC) is one of the preferred separation techniques owing to its good analytical characteristics when coupled to different atomic detectors, particularly with microwave induced plasma (MIP)–optical emission spectrometry (OES) [2, 3]. In at- tempts to overcome the drawbacks associated with the GC speciation of mercury [4], however, different authors have resorted to alternative separation procedures. High-performance liquid chromatography (HPLC) methodologies [5, 6] have been tried with the final aim of establishing a reliable procedure for the speciation of mer- cury compounds. Because HPLC techniques are better suited to the separation of polar species, liquid chro- matography could be advantageous for speciation of organomercury and inorganic mercury. Interfaces for cou- pling HPLC columns with the atomizer are, moreover, rather simple – direct connection of the column exit to the nebulizer of the atomic detector. HPLC methods are, how- ever, significantly less sensitive than GC methods and this is a great limitation for real sample speciation [7]. A pos- sible solution of this lack of sensitivity of HPLC–atomic techniques is on-line derivatization to form a cold vapour (CV) of mercury at the column outlet. SnCl 2 and NaBH 4 are the most popular mercury derivatization reagents to produce Hg cold vapour [8, 9, 10]. Glow discharge (GD) devices have been widely used for the direct analysis of solid materials. Whereas direct- Roberto Martínez · Rosario Pereiro · Alfredo Sanz-Medel · Nerea Bordel Mercury speciation by HPLC–cold-vapour radiofrequency glow-discharge optical-emission spectrometry with on-line microwave oxidation Fresenius J Anal Chem (2001) 371 : 746–752 DOI 10.1007/s002160100972 Received: 13 March 2001 / Revised: 18 May 2001 / Accepted: 29 May 2001 / Published online: 5 October 2001 SPECIAL ISSUE PAPER Dedicated to Professor Dr. Bernd Neidhart on the occasion of his 60th birthday R. Martínez · R. Pereiro · A. Sanz-Medel (✉) Department of Physical and Analytical Chemistry, University of Oviedo, C/ Julián Clavería 8, 33006 Oviedo, Spain e-mail: asm@sauron.quimica.uniovi.es N. Bordel Department of Physics, University of Oviedo, C/ Calvo Sotelo, 33007 Oviedo, Spain © Springer-Verlag 2001