Ammonia-Salicylate Test

Reagents & Equipment

• Colorimeter

• Cuvettes with caps (preferably macro cuvettes)

• Sodium hydroxide, Carolina Biologicals # 889425

• 6% sodium hypochlorite (household bleach)

• Sodium salicylate, Sigma # S2679-100G

• Sodium nitroferricyanide, Sigma #228710-5G

• 1 mL pipette

• 2 x 100 mL volumetric flasks

• Distilled water

Colorimeter set-up

• Wavelength: 625 nm. Red led (colorimeter RGB board)

• Ammonia standard data: Low-range (0 - 2.5 ppm NH₃). Download a data file or prepare your own ammonia standard data

• Before measuring blank the colorimeter against water or a distilled water sample developed with Salicylate reagents as described below

Method

Step 1: Prepare fresh reagents

Hypochlorite solution:

• Fill a 100 mL volumetric flask with 70 mL of DI water.

• Add 1 mL of bleach and 0.5 grams of NaOH and mix until dissolved.

• Fill flask to the 100 mL mark.

Salicylate/catalyst solution:

• Fill a 100 mL volumetric flask with 70 mL of DI water.

• Add 10 g of sodium salicylate, 0.04 g sodium nitroferricyanide and mix until dissolved.

• Add 0.5 g of NaOH to adjust pH to the ~12.0 range.

• Fill flask to the 100 mL mark.

• Transfer solution into a dark, airtight glass bottle for maximum longevity. Prepare fresh weekly due to limited storage life.

Step 2: Colorimetric assay

• Transfer 1 mL of each sample to be tested into a cuvette

• Add 0.25 mL of hypochlorite solution, cap cuvette and invert several times to mix

• Add 0.25 mL of salicylate/catalyst solution, cap cuvette and invert several times to mix

• Let tubes stand for 5-10 mins to develop color.

• Place the cuvette in the colorimeter and click measure. The program will return the ammonia concentration in ppm.