Multi-wavelength spectrophotometric determination of hydrogen peroxide in water with peroxidase-catalyzed oxidation of ABTS

In this study, a new spectrophotometric method was proposed for the measurement of hydrogen peroxide (H2O2) in aqueous solutions. The method was based on the peroxidase (POD)-catalyzed reaction in which 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) was oxidized to form the stable green radical (ABTS+). The generated ABTS+ could be determined spectrophotometrically. The absorbance of the generated ABTS+ at 415 nm, 650 nm, 732 nm and 820 nm were linear with H2O2 concentrations in the range of 0-40 μM. The sensitivities of the proposed ABTS method for H2O2 determination at 415 nm, 650 nm, 732 nm and 820 nm were 6.29 × 104 M-1 cm-1, 2.00 × 104 M-1 cm-1, 2.54 × 104 M-1 cm-1 and 1.89 × 104 M-1 cm-1, respectively. The oxidation of ABTS to generate ABTS+ in POD-catalyzed H2O2 system at pH 6.0 was so fast that the determination time of the ABTS method was as short as 0.5 min. The stoichiometry of the reaction of H2O2 and ABTS in the presence of POD was calculated as nearly 1:2 (1:1.92). The residual absorbance of the generated ABTS+ was also found to be stable within 30 min in natural waters. Low H2O2 concentration in rainwater could be both measured by the ABTS method and the DPD method with high accuracy, however, H2O2 concentration in wastewater contained rhodamine B could only be accurately measured with the ABTS method at 732 nm. Moreover, waste solutions after H2O2 analysis with the proposed ABTS method were non-hazardous towards E. coli.