Protocol for Manual Nitrate Analysis using Superior Stock Nitrate Reductase

      The Nitrate Elimination Co., Inc.
    Green Chemistry for Nitrate Detection
                              Biotechnology People Can Use

  Manual nitrate analysis is carried out by reconstituting freeze-dried AtNaR2 (EC 1.7.1.1) in 25 mM potassium phosphate, 0.1 mM Na2EDTA, pH 7.5, at 22-25°C, to a final concentration of 1 unit per mL. A total of 3 units of NaR are needed to analyze approximately 50 water samples and nitrate standards.

  The unit of enzyme activity is defined as the amount of NaR catalyzing conversion of 1 µmol nitrate to nitrite per min at pH 7.5 and 30°C.

  The Nitrate Analysis Reagent is prepared by bringing the reconstituted AtNaR2 solution (3 units) to 54 mL by mixing with additional phosphate buffer, and the addition of 1.0 mL of 2.82 mM NADH solution, prepared in deionized water. Final volume of the Nitrate Analysis Reagent = 55 mL. This reagent is stable for about 1 hour and should be used immediately after the NADH is added.

  The enzymatic reduction step in the nitrate analysis method is carried out by adding 50 µL of aqueous sample or nitrate standard to 1.0 mL of nitrate analysis reagent, mixing completely with a test tube mixer, and incubating the mixture at 37°C for 15 min in a water bath. Reagent blanks are also prepared by replacing the water sample in the above step with 50 µL of deionized water.  The length of the incubation does not need to be timed exactly and no harm is done by leaving the mixture reacting for up to 2 hours.

  The color development step is carried out by addition of 100 µL of sulfanilamide reagent (10 g per L of ~1.2 N HCl), with vigorous mixing and a 2 min reaction time, which is followed by addition of 100 µL N-(1-naphthyl)ethylenediamine dihydrochloride reagent (1.0 g per L of deionized water) and reacting mixture at 22-25°C or 37°C, for at least 5 min. A longer reaction time is not harmful as long as the absorbance of the samples are read within 1 hour.

   Finally, the absorbance at 540 nm was determined for each sample and nitrate standard using a Hewlett-Packard spectrophotometer (or any colorimeter or spectrophotometer capable of reading absorbance at 540±20 nm is OK.

   A standard curve is generated from the absorbance of nitrate standards prepared in the range of 0.2 to 10 ppm nitrate-N using a certified 1000 ppm nitrate standard (Fisher Scientific Co.). See example of the standard curve in the Results section of NaR-Nitrate Analysis Method.

  The nitrate content of the samples is determined from their absorbance using the nitrate standard curve, which is easily done using EXCEL® or another spreadsheet program.  See example of Nitrate Content in the Results section of NaR-Nitrate Analysis Method.

   Nitrate-free deionized water must be used, and all containers and reaction vessels must also be free of nitrate.  If you have a high background color in all samples, even your control reagent blanks 

NECi Laboratory Nitrate Test Kits (L-NTK) are an easy way to do this manual NaR Nitrate Analysis Method.