View Article Online / Journal Homepage / Table of Contents for this issue

Analyst, June, 1980, Vol. 105, PP. 589-599 589

Determination of Synthetic Colours in Foods Using

Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

High-performance Liquid Chromatography

N. P. Boley, N. G. Bunton, N. T. Crosby, A. E. Johnson, P. Roper and
L. Somers
Deeartment of Industry, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London,
SEl 9NQ

A method is proposed for the extraction, separation, identification and

quantitative measurement of synthetic organic colouring materials in a
wide range of foodstuffs. The colours are extracted with a liquid anion-
exchange resin, and where irreversible binding of colours to the food has
occurred during processing, the colours are released by a preliminary enzyme
digestion prior to extraction. They are then re-extracted from the resin
phase into aqueous solution, followed by clean-up and concentration by
column chromatography on polyamide. The final eluate is concentrated and
examined by high-performance liquid chromatography. For liquid foods
and foods soluble in water, a shortened form of the method can be used. The
method gives recovery values for most colours of 80% or better, and the
reproducibility is within 5 % .

Keywords : Synthetic food colouring materials ; enzymatic digestion ; high-

performance liquid chromatography

Synthetic colours are added to foods to replace natural colour lost in processing, to reduce
batch to batch variation and to produce products with consumer appeal where no natural
colour exists. In recent years food additives in general, and colours in particular, have
increasingly come under investigation for evaluation of their safety in use.l From toxico-
logical data and no-effect levels, acceptable daily intake values (ADIs) for a number of
food colours have been established.2 Because decomposition of some synthetic food colours
is known to occur during processing and on storage, quantitative methods of analysis for
determining the nature and amount of colour present in a food as consumed are essential for
the assessment of the dietary intake levels of these colours. Such methods may well form
the basis of legislative control in the future.
Qualitative methods for the identification of food colours are well established and are
based on paper chromatography3 or thin-layer chr~matography.~Recent advances in
analytical techniques, particularly in high-performance liquid chromatography (HPLC),
have led to the development of methods for the identification of impurities in food colours2
and for the separation of synthetic colours extracted from foods.5
Frequently, experience in using these techniques has shown that extraction of the colouring
matter from the food is incomplete. For quantitative work extraction losses should be
reduced to a minimum. One attempt to overcome this problem was described by Graichen,6
using a liquid anion-exchange resin dissolved in an organic solvent for the extraction stage.
In this method the colours are re-extracted from the resin into aqueous solution and separated
by column chromatography on cellulose ; the separated colours are identified and quantita-
t ively determined by visible-light spect rophot omet ry. Unfortunately , the complete met hod
is very time consuming.
Other workers have used polyamide powder in order to extract colours from water-soluble
foods' and this technique also serves to separate natural from synthetic colouring material,
t o remove sugars, acids, flavouring materials, etc., and to concentrate dilute solutions of
colours.
In this work a method has been developed for the extraction, separation and quantitative
measurement of synthetic organic colouring materials in a wide range of foodstuffs. Synthetic
food colours are extracted from the food with a liquid anion-exchange resin dissolved in
butan-1-01. Where irreversible binding of the colours to the food has occurred during
Crown Copyright.
 View Article Online

690 BOLEY et al. : DETERMINATION OF Analyst, Vol. 105

processing the colours are released by a preliminary enzyme digestion stage prior to extrac-
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

tion.* The colours are next re-extracted from the resin phase into aqueous solution, this
step being followed by clean-up and concentration by column chromatography on polyamide.
The final eluate is concentrated and examined by high-performance liquid chromatography.
The colours are separated by paired-ion chromatography on a reversed-phase column with a
small amount of cetyltrimethylammonium bromide (cetrimide) present in the mobile phase
as counter ion ; they are then determined quantitatively following examination of the column
eluate by visible-light spectrophotometry.

Experimental
Reagents
Use recognised analytica; grade reagents.
Polyamide powder. Grade MN polyamide SC6/CC6 for column chromatography (Camlab
Ltd.).
Sand. Acid washed, 40-100 mesh.
Acetone - water - ammonia solution. Mix 40 ml of acetone, 9 ml of water and 1 ml of
ammonia solution (sp. gr. 0.88). Prepare the mixture fresh daily.
Hydrochloric acid, 0.1 N.
Celite 545 Jilter aid.
Resin-in-butanol solution, 5% V / V . This reagent consists of Amberlite LA-2 resin in
butan-1-01. Shake the resulting solution with 400ml of water containing 19ml of con-
centrated hydrochloric acid in a 2-1 separating funnel. Discard the lower phase.
Sodium chloride solution, 1% m/V.
+
Ammonia solution, 10% V / V . A 1 9 dilution of ammonia solution (sp. gr. 0.88).
Sodium chloride - ammonia solution. Dissolve 10 g of sodium chloride in 300 ml of water,
add 10 ml of ammonia solution (sp. gr. 0.88) and make up to 1 1 with water.
Acetic acid, glacial.
Chloroform.
Heptane,
Diethyl ether.
Phosphate bufer, p H 7.0. Dissolve 2.84 g of disodium hydrogen phosphate and 1.36 g of
potassium dihydrogen phosphate in water and dilute to 1 1.
Acetate bufler, p H 4.6. Add 6.0 ml of glacial acetic acid and 8.2 g of anhydrous sodium
acetate to 100 ml of water and dilute to 1 1.
Enzymes. Papain, lipase, phospholipase C , amyloglucosidase, pectinase and cellulase are
obtainable from Sigma Chemical Co., Poole, Dorset.
Standard colour solutions, 0.1% m/V. Prepare these solutions from standards of known
pure dye content and dilute them to give mixtures of concentration 250 mg 1-l. Store the
solutions in brown bottles or under subdued lighting.

Apparatus
Chromatography column. The column should be approximately 280 mm in length and of
18 mm i.d., and fitted with a PTFE stopcock. Place a plug of glass-wool in the bottom of
the column and add a slurry of 20 g of polyamide powder in 80 ml of water to give a column
approximately 150 mm high. Rinse the walls of the tube with a small volume of acetone
in order to aid the settling of the polyamide and then place sand on top of the polyamide
to form a layer about 6 mm deep.
Buchner funnel, sintered-glass. This should be 200ml in capacity, of No. 3 porosity, and
65 mm in diameter.

HPLC Equipment and Conditions

A Waters Associates 6000A constant-volume pump, fitted with a stop flow injection system
and a 12 cm x 4.6 mm i.d. stainless-steel column packed with 5-pm SAS - Hypersil (Shandon
Southern Instruments Ltd.), operated a t room temperature, is used. Three alternative
methanol - water - cetrimide mobile phases are employed: 77 ml +23 ml + 0.25 g (1) ;
 View Article Online

June, 1980 SYNTHETIC COLOURS I N FOODS USING HPLC 591

80 ml + 20 ml + 0.25 g (2); and 75 ml + 25 ml + 0.25 g (3). The flow-rate of the mobile
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

phase is 1.0 ml min-l. Prepare fresh mobile phase every day and filter it before use through
a 1-pm filter to de-gas and remove particulate matter.
The detector can be a Cecil Instruments CE 515 spectrophotometric detector, 200-800 nm,
operated with a sensitivity of 0.1 a.u.f.s. at: 520 nm for red colours; 480 nm for orange/
brown colours; 430 nm for yellow colours; 640 nm for green/blue colours; and 600 nm for
black colours.
Procedure
A . Aqueous samples and water-soluble foods
Take a known amount (about 10 g) of food and dissolve it in 100 ml of water, warming
if necessary. Next pass the solution through the polyamide column and wash with warm
water (50 ml) and then acetone (15 ml) in order to remove any impurities. Elute the colours
with the minimum volume of acetone - water - ammonia solution, rejecting the eluate until
the colours leave the column.
Evaporate the coloured eluate to dryness on a water-bath under a stream of air, but do
not bake the residue. Re-dissolve it in 1ml of water [or 1 ml of the 77 + +
23 0.25 mobile
phase if Erythrosine BS or Brown FK are present] and retain it for examination by HPLC
according to procedure D.
B. Foods insoluble in water
Transfer a known amount (about l o g ) of food into a pestle and mortar and grind with
10 g of Celite and 10 ml of 0.1 N hydrochloric acid. Transfer the mixture quantitatively to
a sintered-glass Buchner funnel, add 125 ml of chloroform, stir and allow to stand for 5 min.
Then attach the funnel to a water pump, filter the mixture and discard the filtrate. Remove
the funnel from the pump, add 80 ml of resin-in-butanol solution, stir the mixture, allow
it to stand for 10 min and again filter. Repeat the extraction with two further portions of
resin-in-butanol solution. Transfer the combined extracts into a 500-ml separating funnel
and wash them with two 120-ml portions of sodium chloride solution, discarding the aqueous
layer. (Omit this wash when Brown FK is present.) Next add 240 ml of heptane, 100 ml of
ammonia solution and 50 ml of sodium chloride - ammonia solution. Shake the solution
vigorously, allow the phases to separate and collect the aqueous layer. Repeat the extrac-
tion with two further portions of sodium chloride - ammonia solution. Combine the aqueous
extracts, wash with 50ml of diethyl ether and discard the ether layer, then transfer the
aqueous liquid to a 600-ml beaker and warm it on a water-bath under a stream of air for
30 min in order to remove ammonia and residual solvent. Neutralise the solution to pH
6-7 with glacial acetic acid. If Erythrosine BS is present this neutralisation should be
carried out before heating the beaker on the water-bath. Pass the solution through the
polyamide column, and proceed as described under procedure A.
C. Sumfiles where incomplete extraction of colou7 is observed
Transfer a known amount (about 10 g) of food into a 250-ml beaker and add 25 ml of
buffer solution, together with the enzymes selected to hydrolyse the main structural ingredi-
ents (protein, starch, fat, etc.) of the food. For single enzymes use the pH and temperature
conditions outlined in Table VIII. For mixtures of papain and lipase use pH 7.0 buffer and
for mixtures of amyloglucosidase, pectinase and cellulase use pH 4.6 buffer. Incubate the
mixture for 2 h a t a temperature of 30-50 "C, then grind the digested sample with 15 g of
Celite and proceed as in procedure B.
D . Separation and determination of extracted colours by HPLC
Inject a suitable aliquot (1-5 pl) of sample solution obtained from procedure A on to the
column. Record the chromatogram and identify the colours present by reference to
chromatograms obtained following the injection of standard dye mixture using the same
batch of mobile phase. Measure the peak areas and compare them with those given by
standards.
For most separations use mobile phase 1. For colours with long retention times, e.g.,
Ponceau 4R and Black PN, the analysis time can be shortened by using mobile phase 2.
 View Article Online

592 BOLEY et al. : DETERMINATION OF Analyst, Vol. 105

For Chocolate Brown HT use 100% methanol as the mobile phase. Where two colours are
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

incompletely separated, mobile phase 3 may be beneficial. Alternatively, quantitative values

can be obtained by measurement at a wavelength where one colour absorbs weakly, or not
a t all.
Method
Development of the method
Standard solutions were prepared, each containing 25 mg 1-1 of a UK-permitted food colour
or one of the more common colours used abroad. Ten millilitre aliquots of these solutions
were passed through a polyamide column and analysed by use of procedure A (for aqueous
samples and water-soluble foods). The recoveries obtained are shown in Table I, and are
better than SOY0 except for Amaranth, Indigo Carmine and Chocolate Brown HT. Low
recoveries can be caused by the instability of these colours to light, heat and pH changes,
or by incomplete elution from the polyamide column. Some suggestions to minimise these
losses are made later. Further aliquots of standard colour solutions were ground with 10 g
of Celite, extracted with resin-in-butanol solution and analysed following procedure B (for
water-insoluble foods). The recoveries obtained are also shown in Table I and are generally
only slightly lower than those obtained after passage through the polyamide column alone.
TABLE
I
RECOVERIES
OF SYNTHETIC FOOD COLOURS FROM STANDARD SOLUTIONS

-
Recovery, %
Procedure B.
Procedure A . Amberlite LA-2 extraction followed
Polyamide column. by polyamide concentration.
\
Colour 1 2 3 Mean 1 2 3 Mean
Allura Red AC . . .. .. 99 98 93 97 96 92 92 93
Red 2G* . . .. .. .. 97 96 91 95 91 91 94 92
Carmoisine* .. .. .. 92 88 97 92 92 85 88 88
Amaranth* .. .. .. 73 68 61 67 70 79 75 75
Erythrosine BS* . . .. .. 92 89 98 93 94 91 - 93
Ponceau 4R* .. .. .. 97 95 - 96 88 91 - 90
Ponceau SX .. .. .. 96 94 83 91 88 87 91 89
Ponceau 6 R .. .. 100 96 94 97 98 92 94 95
Sunset Yellow F ~ F * .. .. 98 83 95 92 89 93 92 91
Orange RN .. .. .. 98 83 95 92 64 60 58 61
Orange GGN .. .. .. 96 81 93 90 90 94 93 92
Orange G . . .. .. .. 99 83 95 92 91 96 95
-
94
Tartrazine* .. .. .. 87 96 93 92 87 89 88
Quinoline Yellow* .. .. 84 92 90 89 86 86 - 86
Yellow 2G .. .. .. 87 95 94 92 88 86 - 87
Green S* . . .. .. .. 88 89 - 89 70 68 - 69
Patent Blue V* .. .. 94 94 .- 94 94 90 - 92
Brilliant Blue FCF* .. .. 95 95 - 95 96 94 - 95
Indigo Carmine* . . .. .. 64 54 64 61 16 35 - 26
Fast Green FCF . . .. .. 96 91 91 93 96 93 - 95
Black PN* .. .. .. 85 80 79 81 80 82 - 81
Brown FK* .. .. 86 74 - 80 67 67 72 69
Chocolate Brown HT* .. .. 57 58 I
58 41 51 41 44
* Currently permitted in the UK.
Practical di$culties with individual colows
Brown F K . Quantitative analysis of foods containing Brown FK, principally kippers,
necessitated three alterations to the procedures used for the other colours. When a resin-in-
butanol extract containing Brown FK was washed with the sodium chloride solution, the
colour was found to be soluble in the aqueous phase. Hence, this wash must be omitted
when Brown FK is present. Brown FK contains six coloured components, and on extraction
with resin-in-butanol solution the relative amounts of the individual components were
changed and the chromatogram was altered. Quantitative measurements were made by
 View Article Online

June, 1980 SYNTHETIC COLOURS I N FOODS USING HPLC 593

summation of the total areas from all six individual peaks. The final residue, after extrac-
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

tion and concentration, was found to be insoluble in water and was dissolved in 1 ml of
mobile phase before examination by HPLC.
Erythcrosine BS. Erythrosine BS has been shown to interact with some azo colours
including Allura Red AC, Amaranth, Tartrazine and Sunset Yellow FCF.9 The interaction
is dependent on concentration and occurs under alkaline conditions in the presence of light.
When such colour combinations are present in a sample modifications to procedures A and B
are necessary in order to minimise any interaction. Hence, for procedure B, the combined
aqueous extracts obtained following re-extraction of the colours from the resin-in-butanol
were neutralised immediately to pH 6-7 with glacial acetic acid, before heating on a water-
bath to remove residual solvent. Unfortunately, the presence of ammonium acetate, formed
by neutralisation of the ammonia still present, resulted in spreading of the colours on the
polyamide column. This spreading increased the volume of acetone - water - ammonia
solution required in order to elute the colours. The residue obtained was found to be
insoluble in water and was dissolved in 1 ml of mobile phase prior to examination by HPLC.
Green S and Amaranth. Equally, recoveries of Green S and Amaranth by use of procedure
A were increased by neutralisation of the column eluate to pH 7-8 with a few drops of glacial
acetic acid before evaporation to dryness. By reducing interaction with ammonia in this
way the recovery of Green S was increased from 80 to 90% and of Amaranth from 67 to
85%. However, neutralisation of the final eluate prior to evaporation should be employed
only when absolutely necessary. The residue obtained after evaporation will contain salts
which, when dissolved and injected on to the HPLC column, will shorten its working life
and interfere with the chromatographic separation of the colours.
Indigo Carmine. Indigo Carmine was found to be very unstable to light, heat, acids and
alkalis. Quantitative measurements with this compound were variable and low (Table I).
Hence, the determination of this colour should be carried out with as little delay as possible
once analysis has commenced on a sample. For some water-soluble foods it is possible to
determine Indigo Carmine by direct injection of a sample solution on to the HPLC column.
Indigo Carmine has a small retention volume compared with most other colours and was
therefore found to be largely unaffected by the elution of sugars, flavours, etc., present in
the sample solution injected.
Other colours too are partially unstable when exposed to direct sunlight. The content of
Sunset Yellow FCF in an orange drink fell by 14% over a period of 3 months exposure in a
nor t h-f acing window.

TABLE
I1
RECOVERY
OF SYNTHETIC FOOD COLOURS FROM BOILED SWEETS
EXTRACTED BY PROCEDURE A

Amount of
Sample colours added/
number Colours added mg kg-l Recovery, %
1 Quinoline Yellow 25 92,96
Tartrazine 25 89,93
Yellow 2G 25 93,98
2 Sunset Yellow FCF 25 80,76
Red 2G 25 96,99
Amaranth 25 63,64
Carmoisine 25 98,98
3 Ponceau 4R 50 81,84
Black PN 50 82,84
4 Erythrosine BS 100 101,-
5 Green S 12.5 81,92
Brilliant Blue FCF 25 83,95
Patent Blue V 25 83,94

Results
The performance characteristics of the complete method were investigated by the analysis
of a number of foods to which known amounts of colour had been added.
 View Article Online

594 BOLEY et al. : DETERMINATION OF Analyst, Vol. 105

Aqueous Samples and Water-soluble Foods
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

The recovery of added colours from boiled sweets was determined in a series of experiments.
A mixture of 60% sucrose and 40% glucose syrup (42% dextrose equivalent) was prepared,
20 ml water were added and the mixture was boiled until clear and about to solidify. The
viscous mixture was divided into 10-g portions and colours added to the separate portions
as shown in Table 11. Each sample was then dissolved in water and analysed according to
procedure A . The recoveries of added colour are shown in Table 11.
Commercial samples of blackcurrant and lemon jellies, which contain added colours, were
further spiked with standard solutions of colours as shown in Table 111. The jellies were
dissolved in warm water and the colours indicated were added at a level of 25 mg kg-1.
The spiked samples were then examined using procedure A and the recoveries are shown in
Table 111.

TABLE
I11
RECOVERY
OF SYNTHETIC FOOD COLOURS FROM FRUIT JELLIES
EXTRACTED BY PROCEDURE A

Recovery of added
Jelly Colours present Colours added colour, %
Lemon .. .. Tartrazine Red 2G 97,93
Sunset Yellow FCF Carmoisine 93,90
Unidentified natural colours Amaranth 58,63
Blackcurrant .. Carmoisine Quinoline yellow 102,99
Erythrosine BS Yellow 2G 96,96

Similar tests were then carried out by using samples of jams and marmalade known to
contain no added colouring matter. The samples were dissolved in water and colours were
added at a concentration of 50 mg kg-l for Ponceau 4R and Black PN and 25 mg k g l for
the remainder. The samples were then examined using procedure A . Where difficulties
were encountered with samples that contained large amounts of fruit pulp, e.g., strawberry
jam, procedure B was used. With procedure A , an impervious layer of insoluble matter
formed on top of the polyamide column, reducing the rate of flow through the column. The
addition of a layer of glass-wool a t the top of the column largely overcame this problem but
recoveries were usually lower (in the range 30-80y0) owing to adsorption of colour on the
glass-wool and the insoluble residue that did not elute on treatment with the acetone-
water - ammonia solution. Hence, where significant amounts of fruit pulp are present in a
sample it is essential to follow procedure B and extract with resin-in-butanol solution.

TABLE
IV
RECOVERY
OF SYNTHETIC FOOD COLOURS FROM JAM AND MARMALADE

Extraction
Sample Colours added method Recovery, %
Marmalade . . Sunset Yellow FCF Procedure A 95,84
Quinoline Yellow 94,93
Yellow 2G 93,93
Tartrazine 96,93
Strawberry jam . Erythrosine BS Procedure B 94,96
Red 2G 89,97
Carmoisine 93,96
Amaranth 66,67
Ponceau 4R 82,79
Black P N 71,67

Recoveries obtained in the above series of experiments are shown in Table IV. Alternatively,
it is possible to use the enzymic digestion technique described later.
Recoveries of colours added to boiled sweets, jellies, jams and marmalades were found to
be comparable to those obtained for standard colour solutions alone (Table I).
 View Article Online

June, 1980 SYNTHETIC COLOURS I N FOODS USING HPLC 696

Foods Insoluble in Water
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

For these samples procedure B must be used to extract the colours, followed by concentra-
tion and clean-up using procedure A and HPLC analysis according to procedure D.
The technique was first evaluated with samples of sausages and cakes prepared from
known recipes. Two batches of pork and beef sausages were obtained from a commercial
manufacturer using a recipe in which Red 2G was added to produce a theoretical concentra-
tion of 2.6 mg k g l of the colour in the final product; 20-g samples were taken for analysis
and the concentrations of Red 2G found are given in Table V.
TABLE
V
RECOVERY
OF RED2G FROM SAUSAGES EXTRACTED BY PROCEDURE B
Concentration of
Sample Red 2G found/mg kg-1 Recovery, %
Thin beef and pork sausage *. 2.0,2.2 77,85
Thick beef and pork sausage .. 2.3,2.6 88,96

Two batches of sponge cake were prepared according to accepted recipes and standard
solutions of colours were added as shown in Table VI. With sponge 2 a portion of the cake
mixture was examined before cooking. After cooking, the cakes were cooled, weighed and
minced before being subjected to analysis. Allowance was made for changes in the moisture
content. The recovery of added colours is shown in Table VI. Generally, recovery of the
red colours was unsatisfactory and a significant amount of colour was retained in the cake
residue after repeatedly washing with resin-in-butanol solution. This retention suggests
that some form of irreversible binding had taken place between the colour and a constituent
of the cake on baking; the problem is discussed later. Losses of Tartrazine in sponge mixture
2 were probably caused by a bleaching agent present in the batch of commercial flour used.
This effect is shown clearly in Table VI in the low recovery of the colour from the cake mix
before baking. Substitution with a wholemeal flour produced a mixture from which SOY0
of the Tartrazine was recovered.

TABLE
VI
RECOVERY
OF SYNTHETIC FOOD COLOURS FROM SPONGE CAKE
EXTRACTED B Y PROCEDURE B

Amount of
colour added/
Sample Colours added mg kg-l Recovery, yo
'Red 2G 6.2 71,71
Carmoisine 6.2 48,52
Amaranth 6.2 32,36
Sponge 1 Erythrosine BS 7.6 62,-
after baking Ponceau 4R 7.6 43,42
Quinoline Yellow 7.4 80,82
Yellow 2G 7.4 68,88
Tartrazine 7.4 64,82

f:zE
[
Erythrosine BS 9.4 100,-
Tartrazine 23.5 43,-
Patent Blue V 14.5 99,-
[After Erythrosine BS 9.4 64,-
baking Tartrazine 23.5 40,-
Patent Blue V 14.5 91,-

Incomplete Extraction of Colour

For manv foods that are insoluble in water. extraction with resin-in-butanol solution
according t; procedure B was found to remove all the synthetic food colour from the sample,
leaving a colourless residue. For some processed foods, however, synthetic food colour
remained in the sample even on repeated extraction with resin-in-butanol solution. In a
preliminary communications we showed that this problem can be overcome by the use of
 View Article Online

596 BOLEY et al. : DETERMINATION OF Analyst, Vol. 105

enzymes to digest the food prior to extraction. The decision to use enzyme digestion was
more difficult with those foods containing natural as well as synthetic food colours. Resin-
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

in-butanol solution did not extract natural colouring matter from the sample, making it
difficult to perform a visual check on complete extraction of synthetic colours. Such samples
were analysed both with and without enzyme pre-treatment to test for any enhancement of
colour recovery on using procedure C.
TABLE
VII
ENHANCED
RECOVERY OF SYNTHETIC FOOD COLOURS FOLLOWING ENZYME
PRE-TREATMENT USING PROCEDURE c
Colours Colours
extracted by recovered by
procedure B / procedure C/
Sample Enzymes used Colours present mg kg-l mg kg-1
Kippers . . .. Papain, lipase Brown FK 13 29
Salami, Danish . . Papain, lipase Erythrosine BS 2 9
Luncheon meat . . Amyloglucosidase Erythrosine BS 9 11
Chocolate sponge Papain, phospholipase Chocolate Brown HT 6 112
cake Carmoisine 9 15
Plain cake .. Papain Ponceau 4R 14 20
Tartrazine 8 9
Wafer biscuits .. Amyloglucosidase Erythrosine BS 8 21
Strawberry jam .. Cellulase, pectinase,
amyloglucosidase Ponceau 4R 11 19
Fruit pastilles .. Amyloglucosidase Carmoisine 92 116
Green S 2 2
Tartrazine 27 37

In order to test the efficiency of enzyme digestion in releasing synthetic colours bound to
or associated with a food matrix, analyses of processed foods containing known amounts of
added colour are required. The mere addition of synthetic colour to a processed food will
not reproduce the same conditions under which colours are bound to the food matrix. Also,
processing has been shown to cause the decomposition of some colours as a result of heat
instability and reaction with constituents in the food. Under these conditions meaningful
recovery figures cannot be obtained. However, an increased recovery of synthetic food
colour for a number of samples when using enzyme digestion according to procedure C can
be demonstrated. Some typical examples are shown in Table VII. For some foods it was
not always found necessary to hydrolyse all of the main structural ingredients in order to
reach the optimum recovery of colour. The recovery of Erythrosine BS from luncheon meat
was improved following digestion with amyloglucosidase, while hydrolysis of the protein
fraction with papain had no further effect. In contrast, for plain cake no enhancement of
recovery was observed following digestion with a mixture of papain and amyloglucosidase
compared with digestion with papain alone. Some suggestions for the conditions and use
of enzymes are given in Table VIII. Obviously, the colours were associated only with the

TABLE
VIII
CONDITIONSFOR THE ENZYME DIGESTION OF MAJOR FOOD CONSTITUENTS
USING PROCEDURE c
Amount
of
enzyme/ Optimum Temperature/
Substrate Enzyme mg PH "C Examples of use
Protein . . Papain 100 7.0 30 Cake, fish and meat products
Fat . . . , Lipase 50 77 30 Cake, fish and meat products
Phospholipid Phospholipase 10 7.3 30 Sponge cake and egg products
Starch . . Amyloglucosidase 100 4.5 50 Cereals, luncheon meat, jams,
fruit and modified starches
Pectin .. Pectinase 50 4.0 50 Jams and fruit
Cellulose .. Cellulase 50 5.3 50 Jams and fruit
 View Article Online

June, 1980 SYNTHETIC COLOURS IN FOODS USING HPLC 597

cereal starch in luncheon meat and the protein matrix in the plain cake. Not all foods
showed the expected increase in recovery of colour following enzyme pre-treatment . The
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

hydrolysis of sausage meat with papain and amyloglucosidase did not improve on the values
obtained without the use of enzymes. Preliminary enzyme digestion was also used to
solubilise completely some foods, enabling analysis to be continued using the shorter pro-
cedure A .

TABLEIX
SYNTHETIC
FOOD COLOURS FOUND IN LOCALLY PURCHASED FOODS

Analysis Amount of colour

Sample procedures used Colours identified found/mg kg-l
Strawberry drink ., .. .. A, D Sunset Yellow FCF 5
Ponceau 4R 76
Amaranth 20
Blackcurrant drink .. .. .. Amaranth 77
Glucose drink . . .. .. .. Sunset Yellow FCF 28
Tartrazine 25
Boiled sweets : lemon .. .. Sunset Yellow FCF 1
Tartrazine 37
Boiled sweets: lime .. .. .. Tartrazine 25
Green S 11
Boiled sweets : strawbeny .. .. Carmoisine 95
Jelly : blackcurrant .. .. .. Carmoisine 157
Erythrosine BS 18
Jelly: lemon .. .. .. .. Tartrazine 19
Sunset Yellow FCF 1
Marmalade: orange .. .. .. Tartrazine 7
Jam: strawberry .. .. .. Ponceau 4R 41
Fruit pastilles : orange .. .. Sunset Yellow FCF 44
Tartrazine 17
Carmoisine 1
Fruit pastilles: lime . . I .. Brilliant Blue FCF 3
Tartrazine 11
Ice cream: raspberry .. .. .. Ponceau 4R 24
Tartrazine 10
Sausages : pork .. .. .. Red 2G 3
Canned peas . . .. .. .. Green S 4
Tartrazine 8
Brown sauce: fruity . . .. .. Quinoline Yellow 1
Yoghurt : blackcurrant .. .. Carmoisine 5
Instant dessert mix: lemon and lime . . Green S 11
Tartrazine 201
Instant dessert mix : blackcurrant .. Green S 13
Carmoisine 130
Wafer biscuits . . .. .. .. Erythrosine BS 21
Breakfast cereal .. .. .. Tartrazine 158
Sunset Yellow FCF 62
Quinoline Yellow 5
Danish salami .. .. .. Erythrosine BS 16
Ponceau 4R 60
P2t6: lobster .. .. .. .. Erythrosine BS 2
P&t;tB:
liver .. .. .. .. Ponceau 4R 3
Angel cake , . .. .. .. Tartrazine 11
Red 2G 9

Occasionally, problems were encountered during the examination of foods that contained
modified starches by use of procedure A , in that the starch particles reduced the rate of flow
through the polyamide column to an unacceptably low level. This difficulty was overcome
by digestion with amyloglucosidase before addition of the sample solution to the top of the
column. Attention has already been drawn to the problem of samples containing large
amounts of fruit pulp where the shorter procedure A had to be replaced by the longer resin-
in-butanol extraction procedure B. Hydrolysis of a sample of strawberry jam with a mixture
of amyloglucosidase, cellulase and pectinase produced a digest sufficiently clean to be
analysed using procedure A . Thorough washing of the polyamide column with warm water,
 View Article Online

598 BOLEY et al. : DETERMINATION OF Analyst, Vol. 105

before desorption of the colours, was found necessary to prevent reaction products such as
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

sugars and dextrins precipitating out on addition of acetone and so blocking the column.
Washing with 125 ml of warm water was usually found sufficient to prevent this precipitation.
Low recoveries of Green S from blackcurrant pastilles were experienced during experi-
ments in which the enzyme amyloglucosidase was used to break down insoluble starches in
fruit pastilles. No interaction between the colour and the enzyme took place during incuba-
tion. Recoveries of Green S added to orange pastilles varied from 82 to 93%, compared with
only 42% from blackcurrant pastilles. The lower recoveries were thought to be caused by
adsorption of the colour on to the activated carbon present in the blackcurrant pastilles but
absent from the orange flavour. This belief was confirmed by the addition of activated
carbon to the orange pastille at a level of 100 mg k g l , whereupon the recovery of Green S
fell to 43%. For samples containing activated carbon, the full resin-in-butanol extraction
according to procedure B must be employed.

Survey of Locally Purchased Samples of Food

As a test for possible difficulties in applying procedures A-D to a wide range of foodstuffs,
a number of food samples containing added colour were purchased from local retail outlets
and analysed. The samples were examined to determine the nature and concentrations of
added colours, and the results are shown in Table IX. As can be seen, a wide range of
samples covering the major food groups have been tested. The method should be applicable
to all foodstuffs irrespective of the relative proportions of protein, fat or carbohydrate.

Reproducibility of the Method

The repeated analysis of one liquid sample and one solid sample was performed to check
the reproducibility of the method. The results, obtained for a sample of orange drink
containing Sunset Yellow FCF and Tartrazine by use of procedure A , and for a pink portion
of Battenburg cake containing Ponceau 4R by procedure B , are shown in Table X. The
precision obtained was very good, the main source of error being the difficulty in reproducing
the injection of small volumes of extract on to the HPLC column. This reproducibility can
be improved with use of a small, fixed-volume injection loop.

TABLE
X
PRECISION
OF THE METHOD

Amount of colour found/ Standard

mg kg-l Mean/ Variance/ deviation/
Sample Colours present (repeat measurements) mg kg-1 mg kg-1 mg kg-l

i
Sunset Yellow FCF 64.8, 63.7, 64.7, 64.7, 65.3 1.2 1.1
66.2, 64.6, 66.0, 64.2,
66.5, 67.1
Orange drink . . .. Tartrazine 46.1, 46.5, 44.6, 45.8, 45.6 0.5 0.7
46.5, 46.6, 44.9, 45.2,
1 46.1, 44.8
Battenburg cake .. Ponceau 4R 19.3, 20.2, 19.3, 20.2, 19.9 0.3 0.5
20.3, 19.6, 20.7

Conclusions
A method has been developed for the quantitative determination of synthetic food colours
that is applicable to a wide range of foodstuffs. The method includes a novel enzyme
digestion technique to release colour irreversibly bound to some food matrices. The method
was found to have a limit of detection of 1 mg kg-l and gave recovery values for most colours
of 80% or better with a reproducibility within 5%. On gaining experience with the method
it was found possible to complete an analysis within 1 working day. Some of the main
problems encountered in the development of the method have been outlined and suggestions
made to overcome the difficulties encountered. Work is continuing in the laboratory to
improve the analytical procedures for Indigo Carmine and Chocolate Brown HT.
 View Article Online

June, 1980 SYNTHETIC COLOURS I N FOODS USING HPLC 599

We thank the Government Chemist for permission to publish this paper and Williams
Published on 01 January 1980. Downloaded by Otto von Guericke Universitaet Magdeburg on 22/10/2014 09:57:09.

(Hounslow) Ltd. for the gift of dye samples. We also thank the Ministry of Agriculture,
Fisheries and Food for financial support.
References
1. “21st Report of the Joint FAO/WHO Expert Committee on Food Additives,” Technical Report
Series 617, World Health Organization, Geneva, 1978.
2. “Interim Report on the Review of the Colouring Matter in Food Regulations 1973,” Food Additives
and Contaminants Committee, H.M. Stationery Office, London, 1979.
3. Pearson, D. , “The Chemical Analysis of Foods,” Seventh Edition, Churchill Livingstone, Edinburgh,
London and New York, 1976, p. 53.
4. Hoodless, R. A., Pitman, K. G., Stewart, T. E., Thomson, J., and Arnold, J. E., J . Chromatogr.,
1971, 54, 393.
5. Chudy, J., Crosby, N. T., and Patel, I., J . Chromatogr., 1978, 154, 306.
6. Graichen, C., J . ASSOC.Off. Anal. Chem., 1975, 58, 278.
7. Gilhooley, R. A., Hoodless, R. A., Pitman, K. G., and Thomson. J., J . Chromatogr., 1972, 7 2 , 325.
8. Boley, N. P., Crosby, N. T., and Roper, P., Analyst, 1979, 104, 472.
9. Draper, R. E., J . Assoc. Ofi.Anal. Chem., 1975, 58, 614.
Received October 18th, 1979
Accepted December 3rd, 1979