SOP: Determination of Thiamine by the Thiochrome Fluorimetric Method
GIFSQ — Global Institute for Food Safety & Quality
Principle
Thiamine is extracted from the food material with hot dilute acid, and the extract is treated with phosphatase to release thiamine from the bound form. The extract is passed through a base-exchange silicate column to remove interfering substances. Thiamine is eluted from the column and oxidised by alkaline ferricyanide to thiochrome, which is measured fluorimetrically.
Apparatus required
- Boiling water bath
- Incubator (37° C)
- Glass tube column, 10 mm internal diameter and 20 cm length, with a stopcock
- Glass wool
- Buchner funnel
- Volumetric flasks
- Glass-stoppered conical flasks (50 ml)
- Fluorimeter (excitation 360 nm, emission 435 nm) or filter fluorimeter with thiamine primary and secondary filters
- Fluorimeter cells
Reagents
a. 0.2 N HCl: dilute 18 ml of conc. HCl (sp. gr. 1.18) with water to 1 litre.
b. 0.1 N H₂SO₄: dilute 2.8 ml of conc. H₂SO₄ (sp. gr. 1.84) to 1 litre with water.
c. 3% acetic acid: dilute 30 ml of glacial acetic acid (sp. gr. 1.05) to 1 litre with water.
d. 25% (w/v) potassium chloride: dissolve 250 g of KCl in water and make up to 1 litre.
e. Acid potassium chloride solution: dilute 8.5 ml of HCl (sp. gr. 1.18) to 1 litre with 25% KCl.
f. 15% (w/v) NaOH.
g. Alkaline potassium ferricyanide: dilute 3 ml of 1% potassium ferricyanide solution to 100 ml with 15% NaOH. Prepare immediately before use.
h. 2.5 M sodium acetate: dissolve 205 g of anhydrous or 340 g of hydrated sodium acetate in water and dilute to 1 litre.
i. Iso-butanol: redistil iso-butanol in all-glass apparatus; add distilled water and shake until saturated.
j. Enzyme (phosphatase) solution: suspend 6 g of a suitable source of phosphatase in 2.5 M sodium acetate solution and dilute to 100 ml with sodium acetate. Takadiastase of Parke Davis and Co., Clarase of Takamine Laboratories, Polidase of Schwarz Laboratories or Mylase of Wallerstein Laboratories may be used as the source of phosphatase.
k. Activated base-exchange silicate: zeolite (60–90 mesh) and Decalso (60–80 mesh, also called Permutit T) are suitable. Activate as follows: to 100 g of the material, add 200–250 ml of hot 3% acetic acid. After 10–15 min, transfer to a Buchner funnel and apply mild vacuum to drain off the solution. Repeat the acid wash. Then wash with about 250 ml of hot 25% KCl solution followed by hot acetic acid. Finally, wash the material with distilled water till the washings are free from chloride (test the washing for chloride with 1% AgNO₃). Dry at room temperature or any temperature below 100° C.
l. Thiamine stock solution: dissolve 50 mg of thiamine hydrochloride in 0.2 N HCl and dilute to 500 ml with 0.2 N HCl (1 ml = 100 μg).
m. Thiamine working standard solution: dilute 5 ml of the stock solution to 100 ml with 0.2 N HCl. Pipette 5 ml of this solution into a volumetric flask containing 400 ml of acid potassium chloride, and dilute to 500 ml with distilled water (1 ml = 0.05 μg).
Sample preparation (extraction)
Take a known weight of the prepared sample estimated to contain 10 to 30 μg of thiamine
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Add 75 ml of 0.2 N HCl and heat for 30 min in a boiling water bath with occasional shaking
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Cool the extract to room temperature, add 5 ml of freshly prepared enzyme solution, and mix; incubate at 37° C overnight
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Transfer the extract to a 100-ml flask and dilute to 100 ml with water; mix thoroughly and filter
Procedure
Purification on the base-exchange column
Plug the bottom of the glass tube column (above the stopcock) with glass wool, and fill with 6 g of base exchanger suspended in water; allow the water to drain to within 2 mm of the surface of the column bed
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Add 25 ml of the filtrate onto the column and allow to pass through
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Wash the column by passing three successive 10 ml portions of hot water, and discard the washings
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Place 10 ml of acid KCl on the column and allow to pass through; collect the effluent in a 25-ml volumetric flask
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Add another 10 ml portion of acid KCl solution and collect the effluent in the same 25-ml volumetric flask; dilute the contents to the mark with acid KCl solution and mix well
Oxidation and extraction of thiochrome
Pipette 5 ml of the acid potassium chloride eluate into a 50-ml glass-stoppered conical flask; add 3 ml of alkaline ferricyanide solution and mix gently
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Add 15 ml of iso-butyl alcohol and shake vigorously for 90 seconds
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Allow the two phases to separate; remove the lower aqueous layer with a pipette and discard it
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Add 2–3 g of anhydrous sodium sulphate to the iso-butanol phase and shake well
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Into a second 50-ml glass-stoppered conical flask, pipette 5 ml of working standard thiamine solution; carry out the oxidation and subsequent extraction of thiochrome exactly as for the sample
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Prepare a sample blank and a standard blank by treating 5 ml of the sample and standard solutions as above, except adding 3 ml of 15% NaOH instead of alkaline ferricyanide
Measurement of fluorescence
Set the fluorimeter to an excitation wavelength of 360 nm and emission wavelength of 435 nm (or use thiamine primary and secondary filters with a filter fluorimeter)
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Adjust the instrument to zero deflection against 0.1 N H₂SO₄, and 100 against the standard
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Pour the sample into a third fluorimeter cell and measure the fluorescence
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Read the fluorescence of the sample and standard blanks
Calculation
The content of thiamine per gram of the sample in μg is given by the formula:
μg/g = [(A − B)/(C − D)] × (1 × 25)/(5 × V) × (100/wt of the sample)
Where,
- A = reading of the sample solution
- B = reading of the sample blank
- C = reading of the standard
- D = reading of the standard blank
- V = volume of the solution used for adsorption on the column (ml)
- wt of the sample = weight of the sample taken (g)
Precautions
- Prepare the alkaline ferricyanide solution immediately before use.
- Mix gently after adding the alkaline ferricyanide; shake vigorously only after adding iso-butyl alcohol (90 seconds).
- Dry the base exchanger below 100° C and confirm the washings are free from chloride (1% AgNO₃ test).
References
- Bechtel, W.G. and C.M. Hollenbeck, Cereal Chem., 35, 1 (1958).
- McRoberts, L.H., J. Assoc. Offic. Agr. Chem., 43, 47 (1960).
- Conner, R.T. and G.J. Straub, Ind. Eng. Chem., Anal. Edn., 13, 380 (1941).
- Wang, Y.L. and L.J. Harris, Biochem. J., 33, 1050 (1941).
Source flags: the calculation formula is heavily garbled in the extraction; the formula shown matches the garbled layout with the symbols as defined in the book’s own Where key — but the numeric factor “1” cannot be confirmed. From the book’s own stated quantities, the 5 ml standard aliquot contains 5 × 0.05 = 0.25 μg of thiamine, not 1 μg, which would make the result 4× lower than the formula as shown. The printed book MUST be consulted for this formula before publishing. The source prints “18 hl of conc HCl” for reagent (a) — read as 18 ml. The 1% potassium ferricyanide solution used in reagent (g) has no preparation given in the source. “Taka diastase” read as Takadiastase (Parke, Davis and Co.).