Assays of peroxy compounds are commonly performed after chromatographic separation of analysed mixtures. In high-performance liquid chromatography (HPLC), solvent reservoirs are sparged by helium or inline vacuum-degassed in order to control the compressibility of the solvents for efficient pumping. In this study, we investigated the influence of degassing the reaction solution on the light output of the hemin-catalyzed luminol oxidation by various oxidants. We found that, when t-butyl hydroperoxide, hydrogen peroxide, n-butyl hydroperoxide, iodosobenzene and iodobenzene diacetate were used as oxidants, the luminol chemiluminescence was lowered by 50-70% compared with an equilibrated and degassed solution. The opposite effect was observed when dibenzoyl peroxide and 3-chloroperoxybenzoic acid were used as oxidants, as the chemiluminescence increased by approximately 20-30%. The reduced chemiluminescence was explained based on the known role of dioxygen in luminol chemiluminescence. The enhancement of chemiluminescence was rationalized by suggesting an alternative mechanism of luminol oxidation valid for peroxyacids and diacyl peroxides in which the reaction of a peroxyacid anion with the diazaquinone led to light emission with a higher quantum yield than the usual path, which is suppressed by the removal of dioxygen from the reaction solution.
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