SOP: Determination of Riboflavin by the Fluorimetric Method
GIFSQ — Global Institute for Food Safety & Quality
Principle
Riboflavin fluoresces in light of wavelength 440 to 500 nm, and the intensity of fluorescence is proportional to the concentration of riboflavin within a certain range. The vitamin is extracted from the material with hot dilute acids, interference by other substances is eliminated by treatment with potassium permanganate, and the intensity of fluorescence of the resulting solution is measured. In fruit and vegetable products, interfering fluorescing substances are removed with the adsorbent florisil.
Apparatus required
- Boiling water bath
- Volumetric flasks (100 ml)
- Conical flask (100 ml)
- Fluorimeter (excitation 470 nm, emission 525 nm) or filter fluorimeter with riboflavin primary and secondary filters
- Glass adsorption column (16 cm × 1.5 cm) with glass wool (for highly coloured samples)
Reagents
a. 0.2 N HCl: dilute 18 ml of HCl (sp. gr. 1.18) with water to 1 litre.
b. 0.1 N H₂SO₄: dilute 28 ml of H₂SO₄ (sp. gr. 1.84) with water to 1 litre. (Source figure flagged — see note below.)
c. 1 N HCl: dilute 90 ml of HCl with water to 1 litre.
d. 40% NaOH: dissolve 40 g of NaOH in water and dilute to 100 ml.
e. Glacial acetic acid.
f. 3% potassium permanganate: dissolve 3 g of potassium permanganate in distilled water and dilute to 100 ml.
g. 3% hydrogen peroxide.
h. Sodium hydrosulphite (dithionite).
i. Riboflavin stock standard solution: dissolve 25 mg of riboflavin in 3 ml of glacial acetic acid, warming if necessary, and dilute to 1 litre with water (1 ml = 25 μg).
j. Riboflavin working standard solution: dilute 4 ml of the stock standard solution to 1 litre with water (1 ml = 0.1 μg).
k. 2% acetic acid and 20% pyridine in 2% acetic acid (for the florisil adsorption step).
Sample preparation (extraction)
Weigh a sample estimated to contain 5–10 mg of riboflavin into a 100-ml conical flask (source figure flagged — see note below)
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Add 50 ml of 0.2 N HCl and keep in a boiling water bath for 1 hr
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Cool the sample; adjust the pH to 6.0 using NaOH, swirling constantly during the addition of alkali to prevent local areas of high pH
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Add 1 N HCl to bring the pH to 4.5
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Transfer the extract to a 100-ml volumetric flask, make up the volume with water, and filter
Procedure
Oxidation of interfering substances and measurement of fluorescence
Prepare duplicate tubes as follows:
- Tube 1: 10 ml filtrate + 1.0 ml water
- Tube 2: 10 ml filtrate + 10 ml riboflavin working standard
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Add 1 ml of glacial acetic acid to both tubes and mix
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Add 0.5 ml of 3% potassium permanganate to each tube, mix, and allow to stand for exactly 2 min
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Add 0.5 ml of 3% hydrogen peroxide and mix
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Adjust the fluorimeter to an excitation wavelength of 470 nm and emission wavelength of 525 nm (or use riboflavin primary and secondary filters with a filter fluorimeter); adjust to zero deflection against 0.1 N H₂SO₄, and 100 against the contents of tube 2
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Measure the fluorescence of the solution in tube 1
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Add about 20 mg of sodium dithionite to each tube, mix well, and measure the fluorescence within 10 seconds — the readings after addition of sodium dithionite serve as the blank values
Adsorption and elution for highly coloured samples
In a glass adsorption column (16 cm × 1.5 cm), put a wad of glass wool and add florisil to a height of about 8–10 cm
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Wash the column with about 25 ml of 2% acetic acid followed by about 15 ml of water
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Pipette a known volume of the filtrate containing 8–12 μg of riboflavin onto the column
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After the sample has passed through, wash the column with two 15 ml portions of water
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Elute the riboflavin with 25 ml of 20% pyridine in 2% acetic acid and collect the eluate in a 50-ml volumetric flask; add further portions of 20% pyridine in 2% acetic acid and make up to volume
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Use this solution for permanganate oxidation and fluorescence measurement as above
Calculation
Calculate the riboflavin content of the sample using the equation:
μg/g = [(A − B)/((C − D) − (A − B))] × 100/(10 × W)
Where,
- W = weight of the sample (g)
- 100 = volume of the extract (ml)
- 10 = aliquot taken for estimation (ml)
- A = reading of the sample (tube 1)
- B = reading of the sample blank
- C = reading of sample + standard (tube 2)
- D = reading of sample + standard blank
(The 10 ml of working standard added to tube 2 contributes 1.0 μg of riboflavin, which enters the equation as the unity standard-addition factor.)
Precautions
- Riboflavin is light sensitive; avoid exposing riboflavin-containing solutions to strong light.
- Allow the permanganate to stand for exactly 2 min before adding hydrogen peroxide.
- Measure the dithionite blank readings within 10 seconds of mixing.
References
- Hodson, A.Z. and L.C. Norris, J. Biol. Chem., 131, 621 (1939).
- Conner, R.T. and G.J. Straub, Ind. Eng. Chem., Anal. Edn., 13, 385 (1941).
- Kemmerer, A.R., J. Assoc. Offic. Agr. Chemists, 25, 459 (1942).
- Peterson, W.J., D.E. Brady and A.O. Shaw, Ind. Eng. Chem., Anal. Edn., 15, 634 (1943).
- Loy, H.W. Jr., J. Assoc. Offic. Agr. Chemists, 32, 461 (1949).
Source flags: the calculation equation is garbled in the extraction; the formula shown is built from the book’s own Where key (W, 100, 10, A, B, C, D) and its stated tube setup — 10 ml of 0.1 μg/ml working standard adds exactly 1.0 μg, which (C−D)−(A−B) isolates, so the relationship is arithmetically forced by the book’s own quantities. Still to be verified against the printed book. The source prints “5–10 mg of riboflavin” for the sample weight — the standard-addition setup adds only 1.0 μg of standard to a 10 ml aliquot, which suggests the printed figure may be wrong; verify against the printed book before publishing. The source prints “Dilute 28 ml of H₂SO₄” for the 0.1 N acid, while the thiamine method in the same chapter uses 2.8 ml of conc. H₂SO₄ (sp. gr. 1.84) for 0.1 N — 28 ml would give approximately 1 N acid; verify against the printed book (lab-quality issue). No preparation is given in the source for the 2% acetic acid and the 20% pyridine in 2% acetic acid used in the florisil step.