The Scopoletin-HRP Fluorimetric Determination of H(2)O(2) in Seawaters—A Plea for the Two-Stage Protocol

A single solution protocol has been widely used for the fluorimetric determination of H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) in the presence of varying amounts of excess scopoletin, the effect of pH on the rate of reaction between scopoletin and H(2)O(2), 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 H(2)O(2) 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 H(2)O(2) 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.