Riboflavin

Riboflavin (vitamin B₂) is water-soluble and photosensitive. It is present in milk, green leafy vegetables and egg.

Principle
Riboflavin fluoresces at wavelength 440 to 500nm. The intensity of fluorescence is proportional to the concentration of riboflavin in dilute solutions. The riboflavin is measured in terms of the difference in fluorescence before and after chemical reduction.

Materials

» 0.1NH₂SO₄
» Sodium Acetate 2.5M
» Potassium Permanganate 4% (Prepare fresh every time)
» Hydrogen Peroxide 3%
» Riboflavin Standard

Dry standard riboflavin over P₂O₅ in a desiccator for 24h. Dissolve 50mg in 1500mL water and 2.4mL glacial acetic acid in a two liter flask. Warm, cool and make up to volume. Store under toluene in amber bottles in a refrigerator (25mg/mL).
Dilute 20mL of the above riboflavin standard solution to 50mL with water. For working solution, dilute 10mL of the second riboflavin standard solution with water to 100mL. Prepare this dilution fresh. Protect from light. (1mL = 1mg riboflavin)
» Sodium Hydrosulphite (solid)

Procedure

1. Weigh accurately 2-5g of thoroughly mixed and ground sample into a 250mL conical flask.
2. Add 75mL 0.1N H₂SO₄ and mix.
3. Autoclave at 15 lbs for 30 min or immerse the flask in boiling water for 30 min. Shake the flask every 5 min. Let it cool to room temperature.
4. Add 5mL 2.5M sodium acetate solution, mix and let stand for at least 1h. The solution is now at approximately pH 4.5.
5. Transfer to a 100mL volumetric flask and make up the volume with water. While transferring, wash the conical flask completely to transfer the material without any loss.
6. Filter through medium-fast paper such as Whatman No. 2 or No. 4 discarding the first 10-15mL.
7. In test tubes of one inch diameter marked A and B with stirring bars carry out oxidation as follows:
   

   	Low/Blank Tube A	Sample Tube B	High/Blank Tube A	Sample Tube B
   Sample solution (mL)	10	10	10	10
   Standard solution (mL)	1	—	1	—
   Water (mL)	1	2	—	1.0
   *Potassium permanganate (4%) (mL)	0.5	0.5	1.0	1.0
   Time lapse (min)	2	2	4	4
   **Hydrogen Peroxide	0.5	0.5	1.0	1.0
   3% (mL)

   * Stir samples after addition of permanganate. ** Shake after adding peroxide until foaming is negligible.
8. Set the fluorimeter so that glass standard or sodium fluorescein solution gives suitable galvanometer deflection, as directed in the instruction manual of the instrument. Determine fluoroscence of solutions A and B. Do not expose the solution for more than 10 seconds.
9. To solution B in the cuvette, add 20mg sodium hydrosulphite, stir and note the blank fluorescence (C).  Do not use reading (C) taken after colloidal sulphur begins to form.

Calculation

Riboflavin mg/100g =	B – C	x	R	x	V	x 100
	A – B		S		V1

where  
A = reading of the sample plus riboflavin standard
B = reading of sample plus water
C = reading after adding sodium hydrosulphite
R = standard riboflavin added = mg/V₁ of sample solution
V = original volume of sample solution = mL
V₁ = volume of sample solution taken for measurement (10mL)
S = sample weight (g)
In this dilution R = 1; V = 100 and V₁ = 10.

References

1. American Association of Cereal Chemists (1976) (compiled — approved methods committee) AACC Inc St Paul Minn USA 2.