Ammonia-API colorimetric assay

Ammonia in water is commonly measured by the salicylate colorimetric assay. In this assay, the first reaction step involves the conversion of ammonia to monochloroamine by the addition of chlorine. The monochloroamine then reacts with salicylate to form 5-aminosalicylate. Finally, the 5-aminosalicylate is oxidized in the presence of sodium nitroferricyanide (a catalyst) to form a blue-green colored dye that absorbs light at 650 nm.

Reagents

  • API ammonia test kit
  • 1,000 ppm N (1,220 ppm as NH3) standard, Labchem Cat # LC17940-7
  • Distilled water

Equipment

List of vendors and prices: https://sites.google.com/iorodeo.com/colorimeter/equipment

  • Colorimeter with RGB board (use red for measurement)
  • Cuvettes
  • 2 x 50 mL capped tubes
  • 9 x 10 mL capped tubes
  • Pippete pump
  • 2 mL, 5 mL, 10 mL pipettes

Prepare Calibration Solutions

  • Prepare 20 mL of 100 ppm working stock: In a 50 mL tube, add 1.64 mL of 1,000 ppm standard and 18.36 mL of distilled water. Cap and invert to mix.
  • Prepare 20 mL of 10 ppm working stock: In a 50 mL tube, add 2.0 mL of 100 ppm working stock and 18.0 mL of distilled water. Cap and invert to mix.
  • Dilute the 10 ppm working stock as outlined in the Table below

Method

  • To each 5 mL sample, add 8 drops of API Ammonia Test Solution, Bottle #1. Cap the tube and invert to mix.
  • Add 8 drops of API Ammonia Test Solution, Bottle # 2. Cap the tube and shake thoroughly to mix.
  • Wait 5-10 mins for the color to fully develop.
  • Transfer approx. 1mL of each calibration solution to a cuvette.
  • In the colorimeter plotting program, select the Red (625 nm) LED on the standard RGB board and ppm as units. Calibrate the colorimeter with the 0 ppm ammonia 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, there is a second low-range ammonia test using off-the-shelf reagents described here: http://public.iorodeo.com/docs/ammonia/salicylate.html