Effect of Buffer Components on the Determination of Hydrogen Peroxide using Fluorescein Chemiluminescence Reaction

Abstract

A novel chemiluminescent(CL) method for the determination of hydrogen peroxide was developed. The method was based on a fluorescein (FL)-CL reaction catalyzed by horseradish peroxidase at pH 7. O. Effect of four buffer components, citrate, phosphate, imidazole and 2-morpholino-l-propanesulfonic acid (MOPS) on the determination was tested. Citrat e, phosphate and imdazole yielded higher CL intensities than MOPS. Citrate gave a lower detection limit of H₂O₂ than MOPS by a factor of 2.5. Citrate and phosphate were superior to MOPS and imidazole in view of masking transition metal ions which interfered with the determination. A combination of MOPS with EDTA was also effective for precluding the interference from the metal ions.
The CL emission was attribu table to the formation of an excited FL dianion in comparison with the fluorescent and CL spectra of FL. Singlet oxygen also played an impotant role in the formation of the dianion, which was deduced by testing the reactivity of several quenchers and enhancers for active oxygens. Thus, the mechanism of the CL reaction was proposed, in which FL serves dual functions of producing singlet oxygen and of being an energy acceptor from singlet oxygen.