Nitrate-API colorimetric assay

Colorimetric assay for measuring nitrate levels using API test kit. Range: 0-50ppm

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

Absorbance spectrum

  • Reported values in literature [research this]
  • Measured absorbance peak at 540nm
  • Open Colorimeter measurement LED:  520nm


Prepare nitrate calibration solutions: 0-50ppm

  • Prepare a 50 ppm NO3 working stock: 1.129 mL of 4,430 ppm standard in 100 mL volumetric flask. Fill to line with distilled water.
  • Dilute the 50 ppm working stock as outlined below
HTML Table Generator
Nitrate conc. (ppm) 50ppm Nitrate (mL) dist. water (mL)
 0.0  0
 10   1
 20  2
 30  3
 40  4
 50  5


  • 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 60 secs
  • Add 10 drops of reagent from Bottle # 2. Cap the tube and shake thoroughly to mix for 60 secs.
  • Wait at least 5 mins for the color to develop.
  • Make sure the Open Colorimeter has the 520nm LED installed
  • Turn on the Open Colorimeter and select absorbance. Blank the colorimeter with the 0 ppm nitrate solution and measure absorbance of all of the solutions.

Calibration data

From the data, we fit a curve to the concentration as a function of absorbance. 
  "Nitrate": {
    "units": "ppm",
    "led": "520",
    "fit_type": "polynomial",
    "fit_coef": [0.3204, 34.0326, 0.0],
    "range": {"min": 0.0, "max": 1.47}
Nitrate calibration data in Open Colorimeter JSON calibration file format