Direct Determination of Nitrate Using Nitrate Reductase in a Flow System

Abstract

Nitrate was determined using nitrate reductase (NR) in a flow system. A merging zone method was applied in the system in which a zone of NR and that of nitrate in separate streams were merged to react. The NADPH decreased by the enzymatic reaction was detected at 340nm. The length of the reaction coil used for the enzymatic reaction was 250cm. Of the concentrations from 0 to 0.6mм NADPH in a carrier, 0.02mм gave the maximum peak area due to decreased NADPH, suggesting that NR may be inhibited by a coenzyme, NADPH. The buffer of pH 7.5 was found to be optimum in the pH range from 6.5 to 8.0 of the buffer used as a carrier medium. Of the various buffer types(pH 7.5) used as the medium of carriers, piperazine-1, 4-bis(2-ethanesulfonic acid) (PIPES) buffer afforded the maximum peak area. The elevated temperatures of the water bath for enzymatic reaction gave reduced peak areas and the maximum peak area was observed at 32°C. Under the optimum conditions, a linear calibration curve (r=0.996) was obtained in the nitrate concentration range from 5 to 100μм and detection limit (S/N=3) was 1.8μM. The relative standard deviation of the peak area at 20μM nitrate was 4.2% (n=7). The method was applied to the determination of nitrate in samples of natural water. Nitrate content obtained by the present method agreed well with that determined by the JIS method.