The colorimetric assay for nitrite is a single reagent assay based on Gries reaction, whereby nitrite is converted into red azo dye monitored at 540nm. The sequence of reagent (25 mcrL (blue)) and sample (50mcrL (red)) are aspirated into holding coil, forming a single interface (A) within the channel filled with carrier stream of distilled water. Since sample zone is aspirated as second, its leading edge penetrates trough reagent zone, and upon flow reversal the reaction product (yellow) is carried into the flow cell.
The apparatus (B, C) comprises miliGAT pump, LOV with 15cm light path flowtrough cell and 2m long, thermostated holding coil. All lines including the flow cell have 0.8mm I.D.
By using SHC assay protocol detailed on the next page and by using a heated (50 C) holding coil
(HC) the reaction rate was investigated by increasing the time period, within which the reacting mixture was held in the holding coil, while using 100 ppB N nitrite standard (D).
The reaction rate at 50 C is fast enough to allow the nitrite assay to be performed at a rate of 129s/s hr in the range of 0 to 200 ppB N with LOD at 25 ppB N. (E) A higher sensitivity is reached by including a stop time of 10 seconds in the HC and by increasing the injected sample and reagent volumes, as shown on the next passage.