Tutorials

Nitrite-API colorimetric assay

Colorimetric assay for measuring nitrite levels using API test kit. Range: 0-2ppm

Nitrite is measured with the standard Greiss test. In this test, sulfanilamide reagent is converted to a diazonium salt by nitrite (NO2) in the water sample. The diazonium salt is then reacted with NED (N-1-napthylethylene diamine dihydrochloride) reagent to form an azo dye which has a purple/magenta color that is measured at 520-550 nm (suitable for the colorimeter 528 nm green LED).

Absorbance spectrum

Reported values in literature around 540nm
Measured absorbance peak at 530nm
Open Colorimeter measurement LED: 520nm

Materials

Open Colorimeter with 520nm LED board
API nitrite test kit
1,000 ppm N (3,280 ppm as NO₂) standard, Labchem Cat # LC17930-7
Distilled water
Cuvettes
6 x 5 mL capped tubes
2 x 100 mL volumetric flask
1 mL adjustable pipette

Prepare nitrite calibration solutions: 0-2ppm

Prepare 100 ppm NO₂: 3.05 mL of 3,280 ppm standard in 100 mL volumetric flask. Fill to line with distilled water.
Prepare 2.0 ppm NO₂: 2.0 mL of the 100 ppm working stock in 100 mL distilled water in volumetric flask. Fill to the line with distilled water.
Dilute the 2 ppm working stock as outlined below

HTML Table Generator

Nitrite Conc. (ppm)	2ppm Nitrite (mL)	Dist. water (mL)
0.0	0	5
0.4	1	4
0.8	2	3
1.2	3	2
1.6	4	1
2.0	5	0

Method

To each 5 mL sample, add 5 drops of API Nitrite Test Solution
Cap the tube and invert several times to mix.
Wait 5-10 mins for the color to fully 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 nitrite solution and measure absorbance of all of the solutions.