SOP: Determination of Nitrite Nitrogen in Water
GIFSQ — Global Institute for Food Safety & Quality
Principle
Nitrite in water is determined through the formation of a reddish-purple azo dye produced at pH 2 to 2.5 by the coupling of diazotized sulphanilic acid with naphthylamine hydrochloride. The diazotization method is suitable for the visual determination of nitrite nitrogen in the range 0.001–0.025 mg/litre of nitrogen. Colorimetric measurements can be made in the range 0.005–0.05 mg/litre with a 5-cm light path and a green colour filter. The colour system obeys Beer’s law up to 0.18 mg/litre of nitrogen (0.6 mg/litre of NO₂) with a 1-cm light path at 520 nm. The determination should be made promptly on fresh samples to prevent bacterial conversion of the nitrite to nitrate or ammonia.
Apparatus required
- Colorimeter (520 nm)
Reagents
All reagents must be prepared from chemicals which are white in colour.
a. Nitrite-free water: to 1 litre of distilled water, add one small crystal each of KMnO₄ and an alkali such as barium or calcium hydroxide (1 or 2 drops of the alkaline permanganate reagent used for the albuminoid nitrogen determination is also satisfactory); redistil in an all-Corning apparatus, discarding the initial 50 ml of distillate; collect that fraction of the distillate which is free of permanganate. (A yellow colour with the orthotolidine reagent used for the residual chlorine determination indicates the presence of permanganate.)
b. Sulphanilic acid reagent: completely dissolve 0.6 g of sulphanilic acid in 70 ml of hot distilled water, cool, add 20 ml of concentrated HCl and dilute to 100 ml with distilled water.
c. Naphthylamine hydrochloride reagent: dissolve 0.60 g of 1-naphthylamine hydrochloride in distilled water to which 1 ml of concentrated HCl has been added; dilute to 100 ml with distilled water and mix thoroughly. The reagent becomes discoloured and a precipitate may form after 1 week, but it is still usable; discard the reagent when the sensitivity is affected. Store in a refrigerator. Filter before using.
d. 2 M sodium acetate buffer solution: dissolve 16.4 g of sodium acetate (NaC₂H₃O₂) or 27.2 g of its trihydrate (NaC₂H₃O₂·3H₂O) in distilled water and dilute to 100 ml; filter if necessary.
e. Sodium nitrite stock solution: dissolve 0.2463 g of anhydrous sodium nitrite (NaNO₂) in nitrite-free distilled water and dilute to 1000 ml (1 ml = 0.05 mg of N); add 1 ml of chloroform to preserve.
f. Standard sodium nitrite solution: dilute 10 ml of the stock solution to 1000 ml with nitrite-free distilled water (1 ml = 0.0005 mg of N).
g. Aluminium hydroxide: dissolve 125 g of either potassium alum [K₂SO₄·Al₂(SO₄)₃·24H₂O] or ammonium alum [(NH₄)₂SO₄·Al₂(SO₄)₃·24H₂O] in 1 litre of distilled water; heat to 60°C and, with slow mixing, add 55 ml of concentrated NH₄OH; allow to stand for 1 hr; add water and decant the supernatant liquid; wash the precipitate by successive additions of water with thorough mixing and decantation until free from ammonia, chloride, nitrite and nitrate; make up the aluminium hydroxide suspension to 1 litre.
Procedure
If the sample contains suspended matter, add 2 ml of aluminium hydroxide suspension to 100 ml of water, stir, allow to stand for a few minutes, and filter
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Neutralize 50 ml of filtrate or clear sample to a pH of 7
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Add 1 ml of sulphanilic acid (pH should be 1.4 at this point), mix, and allow to stand for 3 to 10 min
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Add 1 ml of naphthylamine hydrochloride reagent and 1 ml of sodium acetate buffer solution; mix well (pH should be 2 to 2.5)
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Allow to stand for 10 to 30 min, and measure the reddish-purple colour at 520 nm, using the reagent blank to set the instrument to 100% transmittance
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Standard curve: dilute 0, 0.25, 0.5, 0.75, 1.5, 2 and 2.5 ml of the standard sodium nitrite solution to 50 ml with nitrite-free distilled water, develop the colour as for the samples, and measure the colour
Calculation
mg/litre nitrite nitrogen = (mg of nitrite nitrogen × 1000) / (ml of sample taken)
Where,
- mg of nitrite nitrogen = nitrite nitrogen read from the standard curve
- ml of sample taken = volume of the water sample taken
mg/litre NO₂ = mg/litre nitrite nitrogen × 3.29
References
- Rider, B.F. & M.G. Mellon, Ind. Eng. Chem., Anal. Edn., 18, 96 (1946).
- Barnes, H. & A.R. Folkard, Analyst, 76, 599 (1951).
- Standard Methods for the Examination of Water and Wastewater, 13th edn., American Public Health Association, Washington D.C., p. 240 (1971).