Flow Injection Tutorial

The determination of nitrate and nitrite is one of the most frequently performed assay on soil and water samples.

The spectrophotometric assay is based on reduction of nitrate to nitrite on Cd column, followed by reaction of nitrite with sulfanilamide. (Gries reaction).The thus produced azo dye is quantified by spectrophotometry @ 540nm.

Optimization of the flow path is more difficult because the void volume of the cadmium column and the tortuous path through it dominates dispersion in the system, while the reduction of nitrate to nitrite is the slowest process of the reaction sequence. Typically , 5cm long column with 2mm internal diameter will have void volume of 500 microliters (assuming 30% packing void). Since the washout of column requires delivery of five void volumes, the minimum flowrate trough the column, to obtain sampling frequency of 2s/min is 2.5mL/min. Unfortunately Cd column is not packed with spherical beads and therefore the void volume is close to 50%. Therefore, by trial and error, typical flowrates per channel in real life constructs, have to be at least 2mL/min per channel.

The attached readout, shows a calibration record of 0, 2, 5, and 8 ppM N as nitrate, followed by a routine run of nitrate assay in samples of soil extracts. The sampling rate is 35 s/hr and detection limit is 1 ppM N.

There are over 335 citations in Hansen’s database dealing with cFI variants of nitrate assay. The focus of research is aimed at improvement or replacement of Cd column, which is the vulnerable component of this assay protocol. So far replacement of Cd column with Zn , enzymes, or UV irradiation has met with a limited success.