Tutorials

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

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

HTML Table Generator

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.