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
| Nitrate conc. (ppm) | 50ppm Nitrate (mL) | dist. water (mL) |
|---|---|---|
| 0.0 | 0 | 5 |
| 10 | 1 | 4 |
| 20 | 2 | 3 |
| 30 | 3 | 2 |
| 40 | 4 | 1 |
| 50 | 5 | 0 |
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
The data plot and JSON calibration data below were generated using the oc-calibration app.