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 (H 2 O 2 ) 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 H 2 O 2 concentrations in the range of 0–40 μM. The sensitivities of the proposed ABTS method for H 2 O 2 determination at 415 nm, 650 nm, 732 nm and 820 nm were 6.29 × 10 4  M −1  cm −1 , 2.00 × 10 4  M −1  cm −1 , 2.54 × 10 4  M −1  cm −1 and 1.89 × 10 4  M −1  cm −1 , respectively. The oxidation of ABTS to generate ABTS + in POD-catalyzed H 2 O 2 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 H 2 O 2 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 H 2 O 2 concentration in rainwater could be both measured by the ABTS method and the DPD method with high accuracy, however, H 2 O 2 concentration in wastewater contained rhodamine B could only be accurately measured with the ABTS method at 732 nm. Moreover, waste solutions after H 2 O 2 analysis with the proposed ABTS method were non-hazardous towards E. coli .