CHEMILUMINESCENCE DETECTION IN LIQUID-CHROMATOGRAPHY BASED ON PHOTOOXYGENATION INVOLVING REACTIVE OXYGEN INTERMEDIATES

A luminol chemiluminescence (CL) detection system for reversed-phase l iquid 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 p hotochemical reactor, which is based on photo-oxygenation induced by t he analytes eluting from the column. In the detector cell CL is genera ted by the microperoxidase (MP-11) catalyzed reaction of the photochem ically produced hydroperoxides and luminol. The experimental parameter s were optimized considering the photochemical reactor and the chemilu minescence detector independently. Optimization of the parameters was directed towards detection of analytes in a gradient reversed-phase ch romatographic system and thus restricted by the eluent composition use d for separation. The solvent composition was not varied freely; in or der to achieve compatibility of the (new) detection system and (gradie nt) RPLC, it is largely determinated by the eluent required to perform the chromatographic separations. The applicability of the optimized s ystem was evaluated for some model analytes and comparisons were made to the CL detection system based on photochemical generation of dioxet anes and to UV-visible absorption detection. Mechanistic implications with respect to the photo-induced production of hydroperoxides in the photochemical reactor were considered and the influences of some known singlet oxygen quenchers and radical chain inhibitors on the signal w ere measured. The detection system was applied to the detection of pol ar pollutants in a gradient reversed-phase chromatographic system, ana lyzing spiked academic solutions and tap-water samples, in the latter case combined with on-line trace enrichment techniques.