Chemiluminescence detection in liquid chromatography based on photo-oxygenation involving reactive oxygen intermediates

Abstract

A luminol chemiluminescence (CL) detection system for reversed-phase liquid chromatography (RPLC), based on on-line photochemical production of organic hydroperoxides is presented. Tetramethylethylene (TME) was used as a reagent for generation of hydroperoxides in a post-column photochemical reactor, which is based on photo-oxygenation induced by the analytes eluting from the column. In the detector cell CL is generated by the microperoxidase (MP-11) catalyzed reaction of the photochemically produced hydroperoxides and luminol. The experimental parameters were optimized considering the photochemical reactor and the chemiluminescence detector independently. Optimization of the parameters was directed towards detection of analytes in a gradient reversed-phase chromatographic system and thus restricted by the eluent composition used for separation. The solvent composition was not varied freely; in order to achieve compatibility of the (new) detection system and (gradient) RPLC, it is largely determinated by the eluent required to perform the chromatographic separations. The applicability of the optimized system was evaluated for some model analytes and comparisons were made to the CL detection system based on photochemical generation of dioxetanes and to UV-visible absorption detection. Mechanistic implications with respect to the photo-induced production of hydroperoxides in the phothochemical reactor were considered and the influences of some known singlet oxygen quenchers and radical chain inhibitors on the signal were measured. The detection system was applied to the detection of polar pollutants in a gradient reversed-phase chromatographic system, analyzing spiked academic solutions and tap-water samples, in the latter case combined with on-line trace enrichment techniques.