The four step reaction (A), which begins with formation of monochloramine, and commences with formation of indophenol blue is slow at room temperature, even when catalyzed by of sodium nitropruside. Therefore, this assay has to be carried out at an elevated temperature.
The miniSIA-1 system (B) was used to apply SHC protocol, at an elevated temperature in a thermostated holding coil, made of 2 m long 0.8mm I.D tubing. The monitoring took place in 6 cm long light path Garth flow cell, kept at a room temperature (21 C). The assay protocol (next page), was designed to assemble the sandwich sequence of hypochlorite, sample and salicylate (100/75/250 microliters).
Using 500ppB N standard, the influence of incubation time at 55 C was investigated (C) followed by study of temperature at incubation time of 5 seconds (D).
Finally, investigation of the Influence of injected sample volume (E) at 55 C and 3 sec incubation time, allowed to optimize the assay protocol shown on the next page.