SOP: Identification of Coal-Tar Colours by Paper Chromatography
GIFSQ — Global Institute for Food Safety & Quality
Principle
Food colours are separated on paper by ascending or circular-zone chromatography in twelve standard solvents, and identified from their Rf values (Table 12-2) or by co-chromatography with standard dyes. Identity is confirmed by comparing the spectrophotometric absorption pattern of the eluted dye with that of the known dye.
Apparatus required
- Whatman No. 1 filter paper (20 × 20 cm, and 11.0 cm circular paper)
- Capillary tube
- White thread
- 2-litre beaker (12.5 cm diameter, 22 cm height), watch glass, polythene sheet, rubber band — or a chromatographic chamber or desiccator
- Two glass plates (lower plate perforated at the centre) and a petri dish (circular zone plate method)
- Spectrophotometer
Reagents
a. Chromatographic solvents 1 to 12:
- 1 ml ammonia (sp. gr. 0.88) + 99 ml water
- 2.5% aqueous sodium chloride (w/v)
- 2% NaCl in 50% ethanol
- iso-Butanol : ethanol : water :: 1:2:1
- n-Butanol : water : glacial acetic acid :: 20:12:5
- iso-Butanol : ethanol : water :: 3:2:2 — to 99 ml of the mixed solvent add 1 ml of ammonia (sp. gr. 0.88)
- 80 g phenol + 20 g water
- Ethyl methyl ketone : acetone : water : ammonia (sp. gr. 0.88) :: 70:30:[fourth component illegible in the available text — printed as a garbled fragment; consult the printed book]
- Ethyl methyl ketone : acetone : water :: 7:3:3
- Ethyl acetate : pyridine : water :: 11:5:4
- Trisodium citrate (2 g), water (95 ml) and 5 ml of ammonia (sp. gr. 0.88)
- HCl (sp. gr. 1.18) : water :: 6.5:30
b. Dye extract: the concentrated dye solution stripped from wool.
c. Standard dyes: solutions of the suspected dyes.
d. 70% alcohol, dilute HCl or NaOH (for confirmation).
Procedure
Ascending paper chromatography
Draw a base line 3/4 inch from and parallel to the bottom of a 20 × 20 cm Whatman No. 1 paper
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Spot a few drops of the unknown dye extract on the line with a capillary tube; spot solutions of the suspected standard dyes alongside
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After drying, curl the paper into a cylinder and join the two ends at two places with white thread
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Place the cylinder in a 2-litre beaker containing about 300 ml of the chosen solvent; cover first with a watch glass, then with polythene, and hold with a rubber band
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Allow the chromatogram to develop until the solvent front has reached 12 cm from the base line; remove and dry
Circular zone plate method
Draw a circle of 3 cm radius in the centre of an 11.0 cm circular Whatman No. 1 paper; cut a “tail” 2 mm wide and 3 cm long starting at the centre
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Spot a few drops of the concentrated dye solution on the circumference of the circle and dry
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Sandwich the paper between the two glass plates (the lower plate perforated to admit the tail); immerse the tail as a wick in the developing solvent in a petri dish, the dish sides supporting the plates
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Allow the chromatogram to develop; remove and dry
Reading and confirmation
Calculate the Rf of each spot and compare with the values in Table 12-2, or compare directly with standard dyes run on the same paper
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Use “double-spotting” to overcome irregularities from food impurities: run three spots — (i) unknown, (ii) unknown + standard control, (iii) control alone
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For spectrophotometric confirmation, build up the spot by repeated addition of sample solution, cut out the resolved spot, extract the dye with a small amount of 70% alcohol, evaporate the alcohol, dilute the nearly neutral residual dye with water, add sufficient HCl or NaOH (for erythrosine) to about 0.1 N, note the absorption pattern in a spectrophotometer, and compare the maxima with those of known dyes determined under similar conditions (Table 12-2 gives the main absorption maxima of several dyes)
Note from the source: Rf is defined as the distance moved by the spot of the food colour divided by the distance moved by the solvent front. The book’s Table 12-2 (Rf values and main spectrophotometric maxima) is only approximate — the values depend on the solvent, the paper, the fibre direction, the developing technique, and the temperature — and serves only as a general guide; consult the printed book for the full table.
Calculation
Rf = Distance moved by the spot of the food colour ÷ Distance moved by the solvent front
Precautions
- Double-spotting (unknown, unknown + standard, standard) is advisable whenever food impurities may distort Rf values.
References
Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.