Pulsed electrochemical detection of thiocompounds following microchromatographic separations

Abstract

Pulsed electrochemical detection (PED) following microchromatographic separations has been applied to the determination of thiocompounds (e.g., methionine, cystamine, glutathione, glutathione disulfide) under typical reversed-phase conditions. Both reduced and oxidized thiol moieties are detected with high selectivity and sensitivity without the need of derivatization. The anodic responses of model thiol redox couples are shown to be based upon oxide-catalyzed anodic oxygen transfer reactions at noble metal electrodes. The mechanism of detection and optimization of PED waveform parameters are studied using cyclic and pulsed voltammetry at rotated disk electrodes. In a comparative study of pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (IPAD), IPAD is determined to be better suited to manage the large oxide-formation (background) currents, which accompany the Faradaic signal of the analyte. IPAD results in more stable baselines, eliminates oxide-induced artifacts, and yields lower limits of detection. The higher frequency waveforms (1 Hz) needed to define the narrow peaks of microchromatographic separations and the fast scan rates (1000 mV s −1) of the IPAD waveform require the use of microelectrodes (≤ 1 mm), which exhibit reduced capacitance effects. Mass detection limits for thiocompounds are typically 0.2–3 pmole for microbore chromatography and 0.1-0.3 pmole for capillary liquid chromatography. The high selectivity of PED for thiocompounds reduces sample preparation and produces simpler chromatograms of complex mixtures, such as liver extracts.