Standard Procedure SP 0005:2005

Method for identifying food dyes in colour-coated chocolate beans

1 Scope

This Standard Procedure identifies food dyes in coloured sweets. It can be used to check that dyes are as stated in the ingredients list. Alternatively, it can be used to identify unknown dyes when these are not specified in the ingredients.

2 Definitions

permitted food dyes
natural or synthetic substances approved for use for colouring foods, to make them look more attractive. Only those tested and proved to be safe are permitted. In the UK and Europe permitted dyes (and other food additives) are identified by E-numbers.

R_f value (Retention factor)
a value that is constant for a given compound in a chromatogram under the same conditions: that is, the same paper (or TLC medium), solvent and temperature. The value allows a chromatogram spot to be identified by comparing its R_f with known values. R_f is calculated as the distance travelled by the compound (measured from the starting position to the centre of the spot) divided by the distance travelled by the solvent in the same time.

3 Principle

Dyes are dissolved from the sweet's surface and absorbed onto wool, to separate them from other soluble ingredients such as sugar. A concentrated solution of the recovered dyes is run against known food dyes on a paper chromatogram. Unknown dyes are identified by comparing the position of spots and/or calculating their R_f values.

4 Apparatus & Reagents

4 boiling tubes
hot water bath (or 400 cm3 beaker)
10 cm3 measuring cylinder
dropper
evaporating basin
stirring rod
tweezers
7 capillary tubes, for spotting the chromatogram
chromatography vessel (or large beaker with cling-film cover)
eye protection
access to fume cupboard
white wool
chromatography paper
6 colour-coated chocolate beans of the same colour
approx 1 mol dm-3 ethanoic acid
approx 6 mol dm-3 ammonia solution CAUTION: corrosive
universal indicator paper
solutions of known food dyes as listed in the ingredients list

5 Procedure

Extracting the dye(s)

Push 10 lengths of white wool (each about 10 cm) into a boiling tube containing 10 cm3 of 1 mol dm-3 ethanoic acid. Heat in a boiling water bath for 5 minutes, removing and shaking gently every half minute.
Allow the tube to cool. Remove the wool, and press dry in a white paper towel.
Meanwhile, just cover six colour-coated chocolate beans (all the same colour) with 1 mol dm-3 ethanoic acid in a boiling tube. Shake gently to dissolve the colour but not the white coating beneath.
Carefully pour off this dye solution into a clean tube. Immerse the dried wool in the dye and heat in the water bath for 5 minutes, stirring occasionally. Remove the dyed wool and rinse it under the tap. Press dry in a paper towel.
In a fume cupboard, put the dyed wool in a clean test tube with 5 cm3 of 6 mol dm-3 ammonia solution. Stir with a glass rod and test a drop on indicator paper. If still acidic (pH<7) add drops of ammonia solution until the solution becomes alkaline.
Heat in the water bath until the dye dissolves out of the wool, which becomes pale. Using tweezers, remove the wool, squeezing it gently to collect as much of the dye solution from it as possible.
Concentrate the dye solution by heating gently in an evaporating basin in a fume cupboard. Stop when a little liquid still remains. If it all evaporates, redissolve in two drops of distilled water.

Paper chromatography

Mark the chromatography paper in pencil, as shown in figure 1.
Use a capillary tube to apply a small spot of the concentrated dye from the sweets at point S - not more than 2mm diameter. Using a clean capillary for each, apply spots of the six known food dyes where shown.
Stand the paper in 1 cm depth of distilled water in a chromatography vessel or large beaker. The water level must be below the line of spots. Seal the vessel and leave the chromatogram to run.
When the water reaches about 1 cm from the top of the paper, remove the chromatogram. In pencil, mark the solvent front (a line across the paper to show how far the water had reached). Hang up the chromatogram to dry.

6 Expression of Results

Compare the colour and position of the spots on the chromatogram. To identify the dye(s) from the sweet, match the spot(s) above position S with those produced by the known dyes.

If more than one spot matches in colour and approximate position, calculate the Rf value for each spot, using the formula:

R_f = d_s / d_w

where

d_s = distance from the base line to the centre of the spot

d_w = distance from the base line to the marked solvent front

Identify the dye(s) from the sweet by matching it to the known food dye with the closest R_f value and same colour. The identity of a spot that does not match within ± 0.1 remains uncertain.

7 Test Report

Your test report should include:

reference to this Standard Procedure;
the colour of the sweet used, and the name and E-number of the food dye(s) identified;
the colour and R_f value of any dye that remains unidentified or uncertain.