Flow Injection Analysis of Hydrogen Peroxide Using Immobilized Horseradish Peroxidase and Its Fluorogenic Substrate 3-(p-Hydroxyphenyl)propionic Acid

Abstract

A highly sensitive, rapid and precise method for the determination of hydrogen peroxide is described which employs flow injection analysis with fluorescence detection. Hydrogen peroxide solution is injected into a water stream, mixed with a solution of 3-(p-hydroxyphenyl)propionic acid, a fluorogenic substrate for horseradish peroxidase. The mixture is then passed through an immobilized horseradish peroxidase column prepared by coupling of the periodate-treated enzyme with aminopropyl glass beads. The fluorescent product in the stream is monitored with a conventional fluorimeter. A sampling rate of 120/h can be attained. The detection limit is 0.5pmol in a 20-μl injection volume. The immobilized enzyme column can be used for a year or longer without loss of the activity.