Application of Flow-ESR Techniques for Mechanistic Study of Biological Radical Reactions: HPLC–ESR Spin-Trapping System for Post-Column Evaluation of the Superoxide Radical Scavenging Activity of Column Eluates

A new high-performance liquid chromatography (HPLC)–electron spin resonance (ESR) spin-trapping system for post-column on-line detection of the superoxide radical scavenging activity of column eluates was developed. A cyclic nitrone-type spin-trapping reagent, 5,5-dimethyl-1-pyrroline- N -oxide (DMPO), was used for detection of the superoxide radical, which was produced by riboflavin-dependent photo-reduction of molecular oxygen in the presence of an electron donor. The reaction conditions were optimized by monitoring the ESR signal intensity of the spin-adduct of DMPO. HPLC–ESR chromatographs were recorded for the standard sample solution, which included three phenolic compounds, gallic acid (Gal), 4-hydroxycoumaric acid (Cou), and caffeic acid (Caf).