Spectrophotometric assay of nitrite is based on Gries reaction, whereby nitrite is converted into red azo dye monitored at 540nm. The reagent , prepared in diluted phosphoric acid is added to sample, injected into carrier stream of distilled water at a confluence point, where streams, each pumped at a rate of 1.5mL/min, merge. The reaction coil, 40 cm long and 0.8mm I.D. was kept at a room temperature. Since the flowrate through reactor (volume 200 microliters) was 50microliters/sec, the incubation time was only four seconds, yet sufficiently long, to produce concentration of the dye that allowed assay to be carried out in a range of 2 to 0.002 ppM of Nitrogen (as nitrite).
The performance of this assay was optimized by:
Minimizing the volume of the reaction coil
Maximizing flow rate
Adjusting the volume of the injected sample
By increasing injected sample volume and absorbance scale the sensitivity of assay increased ten times, while sampling frequency, 100s/hr remained the same. These experiments were carried out long time ago (Ruzicka and Hansen, 1988). Since then, 335 papers on improvement and application of this method have been published according to Hansen’s database.
J. Ruzicka & E.H. Hansen, “Flow Injection Analysis” 2nd ed. J. Wiley, N.Y. 1988