Measuring nitrate with the colorimeter

Nitrate reductase method

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Nitrate reductase method


Reagents & Equipment

  • Colorimeter
  • Cuvettes
  • 5 mL sample bottles or other capped tubes
  • Potassium phosphate (KH2PO4)
  • Potassium hydroxide (KOH)
  • 25 mM EDTA
  • 2 units Nitrate reductase (NR), NECi Cat #
  • NADH
  • Sulfanilamide, Sigma Part #
  • NED
  • (Optional) Distilled water for sample dilutions

Colorimeter set-up

  • Wavelength: 528 nm. Green led (colorimeter RGB board)
  • Nitrate standard data: download a data file or prepare your own nitrate standard data
  • Before measuring blank the colorimeter against water or a distilled water sample developed with API reagent as described below


Step 1: Prepare reagents

  1. Phosphate buffer, pH7.5 (Phosphate buffer (25 mM KH2PO4, 0.025 mM EDTA): Dissolve 3.75 g of potassium phosphate (KH2PO4) and 1.4 g potassium hydroxide (KOH) in 800 mL of distilled water in a 1L volumetric flask. Add 1 mL of 25 mM EDTA and fill to the 1L mark with distilled water.
  2. 2 units/mL Nitrate reductase: Add 1 mL of NECi proprietary enzyme diluent to 2 units of freeze-dried enzyme following the instructions with the enzyme.
  3. 1 mg/ml NADH: Dissolve 0.1 g of NADH (FW=709.4) in 100 mL distilled water. Aliquot and store unused NADH in the freezer.
  4. 1% sulfanilamide: Weigh out 0.15g of sulfanilamide into a small amber bottle. Add 15mL of 3M HCl.
  5. 0.02% NED: Weigh out 2 mg (0.02 g) of NED into an amber bottle. Add 100 mL of distilled water.

Step 2: Prepare enzyme assay mix.

Prepare a “master mix” of enzyme, NADH and phosphate buffer. Make sure you have enough for all of your samples. The Table below can be used for a 10X master mix for 9 samples.

Reagent 1X Mix 10X Mix
Phosphate buffer 890 uL 8.9 mL
1 mg/mL NADH 100 uL 1.0 mL
2 Units/mL NR 10 uL 0.1 mL
Total volume 1000 uL 10.0 mL

Step 3: Assay method

  • Transfer 1 mL of the master mix to clean sample tube e.g. glass test tube.
  • Add 50 µL of water sample and mix thoroughly
  • Incubate at RT for 20-30 minutes.
  • Add 500 µL of 1% sulfanimide and mix
  • Add 500 µL of 0.02% NED and mix
  • Place the sample into a cuvette in the colorimeter and click measure. The program will return the Absorbance and nitrate concentration.

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