FLUOROMETRIC DETERMINATION OF PEROXIDES IN AN HPLC SYSTEM

Abstract

Hemin was used to substitute for horseradish peroxidase (HRP) as catalyst with p-hydroxyphenyl acetic acid (PHPAA) as substrate for post-column detection of H₂O₂ and methyl hydroperoxide (CH₃OOH, MHP) in an HPLC system. In a hemin-catalyzed system, post-column reaction and fluorescence measurement can both be conducted optimally at a pH of ∼10.5. Hydroxymethyl hydroperoxide (HOCH₂OOH, HMHP) and bis(hydroxymethyl) peroxide (HOCH₂OOCH₂OH, BMHP) will rapidly be converted to H₂O₂ in alkaline solution and can also be detected. The HPLC system is optimized for the analysis of H₂O₂ and MHP. Reproducibilities are better than 5% for H₂O₂ and MHP. The detection limits in aqueous solution are 9 nM for H₂O₂ and 200 nM for MHP.

Keywords:

• Fluorescence
• Peroxides
• Hemin
• HPLC
• Postcolumn detection

ACKNOWLEDGMENTS

We are indebted to professor Li Yuanzong, department of chemistry of Peking University, for suggestions and encouragement.