Nitrate
Introduction
The first step in measuring nitrate levels in water is reduction (chemically or enzymatically) of nitrate (NO3) to nitrite (NO2). This is typically carried out using a cadmium-reduction. Other methods using the plant enzyme Nitrate reductase are also used. After reduction, nitrite levels are measured with the Greiss test as the final colorimetric assay step.
Reagents
API nitrate test kit
1,000 ppm N (4,430 ppm as NO3) standard, Labchem Cat # LC17910-7
Distilled water
Equipment
List of vendors and prices: https://sites.google.com/iorodeo.com/colorimeter/equipment
Colorimeter with RGB board (use green for measurement)
Cuvettes
2 x 50 mL capped tubes
9 x 10 mL capped tubes
Pippete pump
5 mL, 10 mL pipettes
Prepare Calibration Solutions
Prepare a 1,000 ppm NO3 working stock: In a 50 mL tube, add 4.51 mL of the 4,430 ppm (1,000 ppm as N) standard and 15.49 mL distilled water.
Prepare a 250 ppm NO3 working stock: In a 50 mL tube add 5.0 mL of the 1,000 ppm standard and 15 mL distilled water.
Dilute the 250 ppm working stock as outlined in the Table 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 30-60 secs
Add 10 drops of reagent from Bottle # 2. Cap the tube and shake thoroughly to mix.
Wait at least 5 mins for the color to develop.
Transfer approx. 1mL of each calibration solution to a cuvette.
In the colorimeter plotting program, select the Green (528 nm) LED on the standard RGB board and ppm as units. Calibrate the colorimeter with the 0 ppm nitrate solution and measure absorbance of all of the solutions in duplicate.
Sample calibration data
To view/use: Download the file and open in the colorimeter plotting software as described here.
In addition to the API test kit, we have also tested two other nitrate tests:
Hach NitraVer test: Nitrate-Hach. Measures 0 - 100 ppm nitrate.
Nitrate reductase test: Nitrate-NR. Measures 0 - 45 ppm nitrate.