SOP: Quantitative Estimation of Added Dyes in Foods

GIFSQ — Global Institute for Food Safety & Quality

Principle

The maximum limit for any permitted coal-tar colour or mixture of colours added to any fruit product is 0.2 g per kg. To estimate the added dye(s) quantitatively, the colour is removed completely from the product without severe heat treatment or strong acids or alkalies (the wool-dyeing technique is undesirable, as repeated boiling is required for complete recovery), and the purified dyes are separated on paper and estimated spectrophotometrically at their wavelengths of maximum absorption.

Apparatus required

• Separating funnel
• Filter
• Paper chromatographic apparatus
• Graduated micro-pipette
• Spectrophotometer

Reagents

a. Higher alcohols: iso-butanol and n-butanol (quinoline may also be used).

b. Weak ammonia solution.

c. Activated alumina (for column chromatography).

d. Polyamide powder (about 10 g per extraction).

e. Dilute acid, 50% (v/v) (for washing the polyamide powder).

f. 5% solution of ammonia in methanol (v/v) (for eluting the synthetic dyes).

g. Paper chromatographic solvents.

Procedure

Extraction

To estimate the added dye(s) quantitatively, remove the colour completely from the product, avoiding any severe heat treatment or the use of strong acids or alkalies — any changes in the dyes during extraction would invalidate the conclusions

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Use any of the following methods to separate the colour from the food:

i. Solvent extraction: the higher alcohols, particularly iso-butanol and n-butanol, are most useful; quinoline may also be used — extract the dye from aqueous solution under acid conditions using the solvent, and remove the dye from the solvent with a weak ammonia solution

ii. Column chromatography: purify the solutions of the extracted dyes by column chromatography using activated alumina

iii. Polyamide powder: to a weak acid extract of colour, or to an acidified liquid sample of food, add about 10 g of polyamide powder, mix thoroughly, and filter — to remove natural dyes, wash the polyamide powder which has adsorbed the dye with dilute acid (50% v/v) till the filtrate is colourless; then elute the synthetic dyes from the polyamide powder with 5% solution of ammonia in methanol (v/v); evaporate the eluate nearly to dryness and use for chromatography

Separation of individual dyes

Make up the purified dye solution to a known volume and spot an aliquot on paper using a graduated micro-pipette

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Develop the chromatogram using a solvent which effects the separation of the individual dyes

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Identify the resolved spots by comparing the shades, Rf values, colour reactions and spectrophotometric curves with those of known dyes

Spectrophotometric estimation of the dyes

Cut the spot from the chromatogram and elute the dye with a known volume of solvent

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Note the absorbance of the dye solution at the wavelength of maximum absorption

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Prepare a calibration curve of the dye using different concentrations; note the concentration of the dye from the calibration curve and calculate the amount of dye present in the product

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The dye content may also be calculated using the extinction coefficient given in Table 12-4

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.