Determination of Carbon-Centered Radicals in Aqueous Solution by Liquid Chromatography with Fluorescence Detection

A simple method to detect subnanomolar to micromolar levels of photochemically generated carbon-centered radicals in aqueous solutions has been developed and optimized. This method Is based on the efficient trapping of radicals by a water-soluble amino nitroxide, followed by derivatization of the trapped products with fluorescamine to produce highly fluorescent adducts. These adducts can be separated by reversed-phase high-performance liquid chromatography and detected fluorometrically. The fluorescent derivatives are stable over a period of days. The detection limit, primarily determined by reagent interferences, ranged from 0.3 to 1 nM per analyte for a 500-µL Injection at a slgnal-to-noise ratio of two. The precision of the method for the determination of adduct concentrations in the 1–10 nM range varied from 2.4 to 8.4% relative standard deviation (n = 6). A direct comparison with electron paramagnetic resonance spectroscopy/spin trapping Illustrates the advantages of our technique. One important feature of the method Is that it permits the simultaneous detection of an array of radicals, as demonstrated through the study of the photochemical production of radicals in a variety of natural water samples and in Suwannee River fulvic acid.