Determination of Nitrate Ion at the Part per Billion Level in Environmental Samples with a Continuous Flow Immobilized Enzyme Reaction

A sensitive procedure for the determination of nitrate based upon the enzymatic reduction of nitrate to nitrite is described, The radical cation of 1,1′-dimethyl-4,4′-bipyridinium dichloride (MV·⁺), which is generated by Na₂S₂O₄ acting upon MV²⁺, reduces nitrate in the presence of the enzyme nitrate reductase. The production of nitrite is monitored with the sulfanilamide N-(1-naphthyl)ethylenediamine dichloride azo dye reaction. A detection limit of 17 ppb NO₃^- using a 1-cm cell in a continuous-flow analyzer was achieved. The major contributor to the noise was derived from the flow system when an optical feedback stabilized photometer was employed. The detection limit using a nonflow system should be about 2 ppb NO₃^- using a 1-cm cell. Use of an immobilized enzyme resulted in quantitative reduction of nitrate and the method is, therefore, relatively free of interferences. Sample concentrations in the range 17 ppb to 7 ppm NO₃^- can be measured. At a concentration of 0.2 ppm, a precision of 1 % can be obtained in a continuous flow analyzer. A variety of real samples were analyzed; correlation with the accepted Cu-Cd reductor method was excellent.