SOP: Determination of Nitrate Nitrogen in Water by the Brucine Method
GIFSQ — Global Institute for Food Safety & Quality
Principle
The reaction between nitrate and brucine yields a sulphur-yellow colour, which is used for the colorimetric estimation of nitrate. The colour system does not obey Beer’s law but gives a smooth curve; it is therefore necessary to develop the colour simultaneously in a series of standards and samples. The intensity of the colour is measured at 410 nm.
Apparatus required
- Colorimeter (410 nm)
- Two 50-ml beakers
Reagents
a. Standard nitrate solution: dissolve 0.7216 g of anhydrous potassium nitrate, KNO₃, in distilled water and make to volume in a 1000-ml volumetric flask (1 ml = 0.1 mg of N). To prepare the working standard solution, dilute 100 ml to 1000 ml with distilled water (1.0 ml = 0.01 mg of N).
b. 0.028 N sodium arsenite (NaAsO₂) solution: dissolve 1.83 g of NaAsO₂ in 1 litre of distilled water; prepare every 6 months. Avoid ingestion.
c. Brucine–sulphanilic acid reagent: dissolve 1 g of brucine sulphate and 0.1 g of sulphanilic acid in approximately 70 ml of hot distilled water; add 3 ml of HCl, cool, and make up to 100 ml. This solution is stable for several months; the pink colour that develops slowly does not affect its usefulness. Avoid ingestion, as brucine is toxic.
d. H₂SO₄ solution: carefully add 500 ml of concentrated H₂SO₄ to 75 ml of distilled water; cool to room temperature before use; keep tightly stoppered to prevent absorption of atmospheric moisture.
Procedure
Standard curve: prepare nitrate standards in the range 0–10 mg of nitrogen per litre by diluting 0, 5, 15, 25, 35, 50, 75 and 100 ml of the working standard nitrate solution to 100 ml with distilled water; pipette 2 ml of each solution, develop and measure the colour as described under colour development below; draw the standard curve by plotting absorbance against concentration
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Pretreatment of sample: if the sample contains chlorine, remove it by adding 0.1 ml of arsenite solution for each 0.05 mg of Cl₂ present, and mix; add one drop in excess to a 50-ml portion
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Colour development: carefully pipette 2 ml of sample containing not more than 10 mg/litre of nitrogen into a 50-ml beaker; add 1 ml of brucine–sulphanilic acid reagent, using a safety pipette
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Into a second 50-ml beaker, measure 10 ml of H₂SO₄; mix the contents of the two beakers carefully by pouring from one beaker to the other, four to six times
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Allow the treated sample to remain in the dark for 10 ± 1 min; while the sample is standing, pipette 10 ml of distilled water into an empty beaker; after 10 min, add the water to the sample and mix as before
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Allow to cool in the dark for 20–30 min; measure the colour of the standards and the sample at 410 nm, setting the blank to 100% transmission
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Run a series of standards with each set of samples; from the standard curve, note the concentration of nitrate nitrogen in the sample
Calculation
mg/litre nitrate nitrogen = (mg of nitrate nitrogen × 1000) / (ml of sample taken)
Where,
- mg of nitrate nitrogen = nitrate nitrogen in the sample read from the standard curve
- ml of sample taken = volume of the water sample taken
mg/litre NO₃ = mg/litre nitrate nitrogen × 4.43
Note: the book states the method is suitable for determining nitrate nitrogen from “1 to 10 mg/ml”; this is carried as printed but flagged — the standard curve and all working dilutions are in mg/litre, and “mg/ml” here reads as a source error.
References
Noll, C.A., Ind. Eng. Chem., Anal. Edn., 17, 426 (1945).