Determination of Nitrite in Waters by Microplate Fluorescence Spectroscopy and HPLC with Fluorescence Detection

A selective and versatile fluorescence spectroscopic method for the determination of nitrite in waters has been developed. Nitrite reacts in the presence of mineral acids with the nonfluorescent N-methyl-4-hydrazino-7-nitrobenzofurazan forming N-methyl-4-amino-7-nitrobenzofurazan, which can be detected by fluorescence spectroscopy with an excitation maximum at lambda = 468 nm and an emission maximum at lambda = 537 nm in acetonitrile. Three new methods based on this reaction have been developed: Direct fluorescence spectroscopy, HPLC/fluorescence, or HPLC with UV/vis detector may be selected as detection techniques. On microplates, high-throughput fluorescence spectroscopy is achieved, while HPLC/fluorescence provides lower limits of detection, and HPLC with UV/vis detection enables evaluation of the reaction with standard instrumentation. Different water samples were investigated using all detection modes, and a photometric standard procedure was successfully employed to validate the new methods with an independent technique.