Berthelot reaction, salicylate analog of indophenol blue. Ammonia reacts with hypochlorite to form chloramine, which couples with two non-para-substituted phenols. Citrate and tartrate are included to avoid precipitation of Ca, Mg, and other hydroxides.
Forster JC. 1995. Soil nitrogen. In: Alef K and Nannipieri P (eds.) Methods in Applied Soil Microbiology and Biochemistry. Academic Press, San Diego. pp. 79-87.
Kempers AJ, Kok CJ. 1989. Re-examination of the determination of ammonium as the indophenol blue complex using salicylate. Anal. Chim. Acta. 221:147-155.
Verdouw H, van Echteld CJA, Dekkcrs EMJ. 1977. Ammonia determination based on indophenol blue formation with sodium salicylate. Water Res. 12:399-402.
-The procedure below most closely matches Forster 1995.
Reagent A: in 100 ml water
- 6.5 g sodium salicylate
- 5 g sodium citrate
- 5 g sodium tartrate
- 0.025 g sodium nitroferricyanide (sodium nitroprusside)
This reagent is somewhat sensitive to light. It may be stored cold. Discard if dark.
Reagent B: in 100 ml water
- 6 g sodium hydroxide
- 2 ml bleach (5.25% sodium hypochlorite) or equivalent
†Quantities in reagent preparation need not be exact.
In semimicro cuvets mix sample, then A, then B, in the appropriate amounts. Cap and invert to mix. Read absorbance at 650 nm against a reagent blank after 50-60 minutes.
- For 1-20 ppm NH use 40 µl sample, 500 µl A, 500 µl B.
- For 1-10 ppm. use 80 µl sample, 500 µl A, 500 µl B.
- For 1-5 ppm, use 140 µl sample, 400 pl A, 400 µl B. (to ~0.1)
- For less than 1 ppm* use 600 µl sample, 150 µl A, 150 µl B. Working lower limit is approximately 0.025 ppm N.
- For 5-60 ppm*, use macrocuvets, 45 µ1 sample, 1500 µl ½ x A, 1500 µl ½ x B. (lower limit ≈ 1 ppm)
Primary amines e.g. amino acids) and certain other N compounds may interfere slightly when present in large amounts (more than 10-20 times the ammonia concentration). It is usually not a problem with soil extracts and water samples. For reducing such interference substitute sodium 2-phenylphenolate for sodium salicylaic (Rhine et al Soil Sci. Soc. Am. J. 62:473-480).
*Note: When using < 1 ppm range with high Ca ⁄ Mg samples (e.g. soil extracts) use 700 µl sample, 300 µl 2.5 × A**, 200 µl B.
**2.5 × A: in 100 ml, 17 g sodium salicylate, 12.5 g each sodium citrate and tartrate, 62 mg sodium nitroprusside.