Spectrophotometric method for superoxide anion radical detection in a visible light (400-780 nm) system

A method was developed that used hydroxylamine hydrochloride as a probe in a superoxide anion radical-generating visible light system for determining superoxide anion radicals. An Azure I solution with hydroxylamine hydrochloride was illuminated, after which a ferric iron solution was added to the sample solution and the remaining hydroxylamine hydrochloride in solution reduced from ferric to ferrous ions. Then, 1,10-phenanthroline solution was added and spectrophotometrically measured at 510 nm, which indirectly indicated the hydroxylamine hydrochloride content. The yields of superoxide anion radicals were indirectly expressed by the hydroxylamine hydrochloride decrement. Under optimal experimental conditions, the linear range was 0.0-1.5 × 10^-5 M and the limit of detection and limit of quantification were obtained to be 8.37 × 10^-7 and 2.54 × 10^-6 M with an R² of 0.9993. The method was simple and feasible and could be used for the stable measurement of superoxide anion radicals produced by photosensitizers that produce color under acidic conditions in visible light systems.