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Methods Protoc. 2018, 1(1), 4;

The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol

1,2,* , 3,4,* and 3
Key Laboratory of Global Change and Marine-Atmospheric Chemistry (GCMAC), Third Institute of Oceanography (TIO), State Oceanic Administration (SOA), Xiamen 361005, Fujian, China
State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China
Research Center for Environmental Changes, Academia Sinica, Nankang, Taipei 115, Taiwan
Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, Norfolk, VA 23529, USA
Authors to whom correspondence should be addressed.
Academic Editor: Fernando Albericio
Received: 31 October 2017 / Revised: 26 November 2017 / Accepted: 27 November 2017 / Published: 1 December 2017
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A single solution protocol has been widely used for the fluorimetric determination of H2O2 in natural waters by its bleaching of the fluorescing scopoletin in the presence of the enzyme horseradish peroxidase (HRP). In this protocol, the reaction between scopoletin and H2O2 in the sample and the subsequent internal additions, and the measurements of the fluorescence are all carried out at a single pH in a fluorometer cell. It is found that this protocol is prone to four sources of possible error. The variability in the reaction stoichiometry between scopoletin and H2O2 in the presence of varying amounts of excess scopoletin, the effect of pH on the rate of reaction between scopoletin and H2O2, the photobleaching of scopoletin, and the de-activation of HRP. These possible sources of error can be circumvented in a two-stage protocol in which the reaction between H2O2 and scopoletin is carried out immediately upon sampling at a pH of 7, and the measurement of the fluorescence is carried out later on at a pH of 9. It should be the protocol of choice. Furthermore, in the two-stage protocol, after the initial reaction between H2O2 and scopoletin, the sample may be stored at room temperature for six days and at 4 °C for at least a month before its fluorescence is measured. This option can significantly reduce the logistics in the field. View Full-Text
Keywords: hydrogen peroxide; fluorimetric determination; scopoletin; horseradish peroxidase hydrogen peroxide; fluorimetric determination; scopoletin; horseradish peroxidase

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Wu, M.; Wong, G.T.F.; Wu, Y.-C. The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol. Methods Protoc. 2018, 1, 4.

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