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REDUCTION OF NITRATE THROUGH THE USE OF NITRATE REDUCTASE FOR THE SMARTCHEM AUTOANALYZER
Citation:
ALMASSALKHI, B. AND K. KELTY. REDUCTION OF NITRATE THROUGH THE USE OF NITRATE REDUCTASE FOR THE SMARTCHEM AUTOANALYZER. Presented at Central Regional Meeting of the American Chemical Society 2007, Covington, KY, May 20 - 23, 2007.
Description:
The standard method for the determination of nitrate in drinking water, USEPA Method 353.2 Determination of Nitrate-Nitrite by Automated Colorimetry, employs cadmium as the reductant for the conversion of nitrate to nitrite. The nitrite is then analyzed colorimetrically by way of the Griess reaction. This procedure is not only tedious, but the by products of the cadmium column used is hazardous to the environment. However, there is another alternative: Superior Stock Nitrate Reductase or YNaR1 (The Nitrate Elimination Co. of Lake Linden, Michigan). This enzyme combined with NADH as the reductant, can replace the cadmium column reduction thereby eliminating the need to dispose of toxic cadmium waste. Initial experiments were performed with a Cary 50 UV-VIS and a 37°C water bath to emulate the temperature settings for the final instrumentation, a SmartChem discrete colorimetric auto analyzer (Westco Scientific, Danbury, CT). Early results proved successful in reducing the nitrate to nitrite. However, it was found that the NADH interfered with the Griess color reaction. An optimization study was then done to determine which concentration of NADH and YNaR1 is needed to reduce the nitrate while, at the same time, providing the least interference for the color reaction. It was found that increasing the concentration of NADH, to 3-4x the highest concentration of nitrate in the sample, will guarantee that there will be enough NADH to propel the reaction. Using this information, a new method was implemented onto the SmartChem, NO3R, and the enzyme was tested at different concentrations. As the concentration was increased from 0 to 6 µg/mL YNaR1 the reduction peaked at the 4 µg/mL or 2x the recommended concentration. Using a students T-test, this nontoxic way of reducing nitrate was found and proven to be successful within an 80% confidence limit of the standard USEPA Method 353.2.