Nitrate-API colorimetric assay

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

Materials

• Open Colorimeter with 520nm LED board
• API nitrate test kit
• 1,000 ppm N (4,430 ppm as NO3) standard, Labchem Cat # LC17910-7
• Distilled water
• Cuvettes
• 6 x 10 mL capped tubes
• 1 x 100 mL volumetric flask
• 1 mL adjustable pipette

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

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 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

{
  "Nitrate": {
    "units": "ppm",
    "led": "520",
    "fit_type": "polynomial",
    "fit_coef": [0.3204, 34.0326, 0.0],
    "range": {"min": 0.0, "max": 1.47}
  }
}