Nitrate

Introduction

The first step in measuring nitrate levels in water is reduction (chemically or enzymatically) of nitrate (NO3) to nitrite (NO2). This is typically carried out using a cadmium-reduction. Other methods using the plant enzyme Nitrate reductase are also used. After reduction, nitrite levels are measured with the Greiss test as the final colorimetric assay step.

Reagents

  • API nitrate test kit

  • 1,000 ppm N (4,430 ppm as NO3) standard, Labchem Cat # LC17910-7

  • Distilled water

Equipment

List of vendors and prices: https://sites.google.com/iorodeo.com/colorimeter/equipment

  • Colorimeter with RGB board (use green for measurement)

  • Cuvettes

  • 2 x 50 mL capped tubes

  • 9 x 10 mL capped tubes

  • Pippete pump

  • 5 mL, 10 mL pipettes

Prepare Calibration Solutions

  • Prepare a 1,000 ppm NO3 working stock: In a 50 mL tube, add 4.51 mL of the 4,430 ppm (1,000 ppm as N) standard and 15.49 mL distilled water.

  • Prepare a 250 ppm NO3 working stock: In a 50 mL tube add 5.0 mL of the 1,000 ppm standard and 15 mL distilled water.

  • Dilute the 250 ppm working stock as outlined in the Table below

Standard Curves

Method

  • To each 5 mL sample, add 10 drops of reagent from Bottle # 1 to the tube, cap and invert several times to mix

  • Vigorously shake Bottle # 2 for 30-60 secs

  • Add 10 drops of reagent from Bottle # 2. Cap the tube and shake thoroughly to mix.

  • Wait at least 5 mins for the color to develop.

  • Transfer approx. 1mL of each calibration solution to a cuvette.

  • In the colorimeter plotting program, select the Green (528 nm) LED on the standard RGB board and ppm as units. Calibrate the colorimeter with the 0 ppm nitrate solution and measure absorbance of all of the solutions in duplicate.

Sample calibration data

  • To view/use: Download the file and open in the colorimeter plotting software as described here.

In addition to the API test kit, we have also tested two other nitrate tests:

  • Hach NitraVer test: Nitrate-Hach. Measures 0 - 100 ppm nitrate.

  • Nitrate reductase test: Nitrate-NR. Measures 0 - 45 ppm nitrate.