SOP: Determination of Folic Acid by the Colorimetric Method
GIFSQ — Global Institute for Food Safety & Quality
Principle
Folic acid is extracted from food samples using a mild alkaline buffer, oxidised with permanganate, and the resulting amine is diazotized. The diazotized compound is coupled with N-(1-naphthyl)ethylenediamine, and the colour developed is determined at 550 nm after extraction into iso-butyl alcohol.
Apparatus required
- Volumetric flasks (100 ml)
- Assay tubes (in duplicate)
- Centrifuge
- Colorimeter or spectrophotometer (reading at 550 nm)
Reagents
a. Dibasic potassium phosphate (K₂HPO₄) solution: dissolve 60.61 g of K₂HPO₄ in water and make up to 2 litre.
b. Potassium permanganate solution: take 0.4 g of KMnO₄ in a 100-ml volumetric flask and dilute to volume with water.
c. Sodium nitrite solution: take 2 g of NaNO₂ in a 100-ml volumetric flask and make up to volume with water.
d. 5 N HCl.
e. Ammonium sulphamate solution: dissolve 5 g of ammonium sulphamate in water and make up the volume to 100 ml. (The salt’s formula is garbled in the source print — see note below.)
f. N-(1-naphthyl)ethylenediamine dihydrochloride solution: place 0.1 g of the substance in a 100-ml volumetric flask and dilute to volume with water.
g. Sodium chloride.
h. Iso-butyl alcohol.
i. Stock folic acid standard solution: dissolve 50 mg of folic acid in water with the help of 2 ml of NH₄OH, and make up the volume to 100 ml with water (1 ml = 500 μg).
j. Working folic acid standard solution: dilute an aliquot of the stock standard solution with K₂HPO₄ solution (reagent a) to give a concentration of 10 μg/ml.
Sample preparation
Place a known amount of the sample estimated to contain about 100 mg of folic acid in a 100-ml flask (“a cet” read as “about” — source figure flagged, see note below)
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Add about 50 ml of K₂HPO₄ solution and heat the mixture to a temperature not above 60° C, swirling until the sample is properly dispersed
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Cool to room temperature and make up the volume to 100 ml with K₂HPO₄ solution
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Filter or centrifuge the solution
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Transfer an aliquot of the clear solution estimated to contain about 1 mg of folic acid to a 100-ml volumetric flask and dilute to volume with K₂HPO₄ solution
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Use this solution for colour development and estimation
Procedure
Prepare duplicate assay tubes as follows (volumes in ml):
- Tube 1: 5.0 sample, no working standard, no water, 1.0 potassium permanganate solution
- Tube 2: 5.0 sample, 2.0 working standard, no water, 1.0 potassium permanganate solution
- Tube 3: 5.0 sample, no working standard, 1.0 water, no permanganate
- Tube 4: 5.0 sample, 2.0 working standard, 1.0 water, no permanganate
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Add 1 ml of sodium nitrite solution and 1 ml of 5 N HCl to all tubes; mix and allow to stand for 2 min
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Add 1 ml of ammonium sulphamate solution and mix with swirling
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Add 1 ml of N-(1-naphthyl)ethylenediamine dihydrochloride solution, mix, and allow to stand for 10 min
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Add 1 g of sodium chloride and 10 ml of iso-butyl alcohol; shake vigorously for 2–3 min
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Separate the iso-butyl alcohol layer by centrifugation and remove about 9 ml of the clear supernatant layer
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Read the colour of the iso-butyl alcohol at 550 nm within 25 min, using iso-butyl alcohol as the blank
Calculation
Calculate the quantity of folic acid in the sample preparation using the expression:
folic acid = C × 2 × (A₁ − A₃) / [(A₂ − A₄) − (A₁ − A₃)]
The expression gives the folic acid in the 5 ml sample aliquot, in the same units as C.
Where,
- C = concentration of the working standard of folic acid (mg/ml, as given in the source — note the working standard as prepared is 10 μg/ml = 0.01 mg/ml)
- A₁, A₂, A₃, A₄ = the absorbance of tubes 1, 2, 3 and 4 respectively
- 2 = ml of working standard added to tubes 2 and 4
Precautions
- Do not heat the sample mixture above 60° C during extraction.
- Read the colour at 550 nm within 25 min of colour development.
- Allow the diazotization step to stand for the full 2 min, and the coupling step for 10 min.
References
- Hutchings, B.L., E.L.R. Stokstad, J.H. Booth, J.H. Mowat, O.W. Waller, R.B. Angier, J. Semb and Y. Subba Row, J. Biol. Chem., 168, 705 (1947).
- Kaselis, R.A., W. Liebermann, W. Seaman, J.P. Sickels, E.I. Stearns and J.T. Woods, Anal. Chem., 23, 746 (1951).
- Schiaffino, S.S., J.M. Webb, H.W. Loy and O.C. Kline, J. Am. Pharm. Assoc., 48, 236 (1959).
Source flags: the calculation formula is fully garbled in the extraction; the formula shown is the standard-addition relationship built from the book’s own tube setup (tubes 1–4) and its Where key (C, A₁–A₄). The sample figure reads “a cet 100 mg” (“a cet” read as “about”); with 100 mg in the first 100-ml flask, an aliquot containing about 1 mg is internally coherent with the tube setup (5 ml ≈ 50 μg against a 20 μg standard addition) — verify the absolute levels against the printed book before publishing. The salt’s formula in reagent (e) is garbled in the source print (“(NH4)NHe2SO”); the standard salt is ammonium sulphamate, NH₄SO₃NH₂ — flagged for verification.