Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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on account of their low tinctorial power and instabil-

ity to further oxidation.

0031 Carbon black is an insoluble pigment derived from

the controlled combustion of plant material and is

usually converted into a liquid suspension by milling

into a carrier such as sugar syrup or fat. In this form

it is used in the manufacture of black sugar confec-

tionary.

Analysis of Natural Colors

0032 Analysis of natural colors as individual pigments is

really only possible using chromatographic tech-

niques, although the use of tristimulus colorimetry is

valuable in defining and maintaining the exact visual

specification of a color in a foodstuff. Extraction of

pigments requires specific techniques, e.g., acidified

alcohol/water mixtures for anthocyanins, ethyl acet-

ate or dichloromethane for carotenoids. Most ana-

lyses are nowadays carried out by high-performance

liquid chromatography (HPLC) on reverse-phase

columns. For anthocyanins, water/alcohol gradients

at pH 1.5 are suitable. For carotenoids and chloro-

phyls, mixtures of dichloromethane, acetonitrile, and

tetrahydrofuran are often used. Detection must be in

the visible spectrum – diode array detectors can be

helpful in confirming peak identity. Isolation of

standards is often a major difficulty since few are

commercially available.

0033 In processed food the added pigments may be de-

graded or oxidized during processing, or may become

so tightly bound to the food matrix that their total

recovery is impossible. For these reasons the quanti-

tation of added anthocyanins or carotenoids to a

foodstuff is only approximate at best, especially if

these pigments have themselves been added as

naturally occurring mixtures without a defined

composition. If simple synthetic b-carotene has

been added to a food, it can be extracted by sa-

ponification under nitrogen followed by ether ex-

traction and HPLC, and then quantified with

reference to an external standard. However, the quali-

tative identification of pigment origin can be a power-

ful tool. For instance, fruit desserts colored with

grapeskin or with black carrot extracts can be readily

identified from each other, and likewise sauces

colored with paprika extracts or b-carotene can be

easily distinguished. Caramels and phenolic oxida-

tion products cannot be analyzed as single chemical

entities due to their heterogeneous nature, although

the presence of byproducts such as hydroxymethyl-

furfural may be useful for the indirect assessment

of caramel addition. New techniques such as capillary

zone electrophoresis show promise for the analysis

of charged materials such as the class IV E150(d)

caramels.

See also: Antioxidants: Synthetic Antioxidants;

Browning: Nonenzymatic; Caramel: Properties and

Analysis; Carotenoids: Occurrence, Properties, and

Determination; Chlorophyl; Chromatography: High-

performance Liquid Chromatography; Curing; Retinol:

Physiology

Further Reading

Blake CJ (1982) Determination of Natural Colours in

Foods. Scientific and Technical Survey no. 130. Leather-

head: Leatherhead Food Research Association.

Coulson J (1980) Naturally occurring colouring matters

for foods. In: Walford J (ed.) Developments in Food

Colours, vol. 1, pp. 189–218. London: Elsevier.

Henry BS (1996) Natural food colours. In: Hendry GAF

and Houghton JD (eds) Natural Food Colorants, 2nd

edn, pp. 40–79. London: Blackie.

Knewstubb CJ and Henry BS (1988) Natural Colours – A

Challenge and an Opportunity, pp. 179–186. London:

Institute of Food Science and Technology.

Lea AGH (1988) HPLC of natural pigments in foodstuffs.

In: Macrae R (ed.) HPLC in Food Analysis, 2nd edn,

pp. 277–333. London: Academic Press.

Markakis P (1982) Anthocyanins as Food Colours.

London: Academic Press.

Taylor AJ (1984) Natural colours in food. In: Walford J

(ed.) Developments in Food Colours, vol. 2, pp. 159–

206. London: Elsevier.

Timberlake CF and Henry BS (1986) Plant pigments as

natural food colours. Endeavour 10: 31–36.

Properties and Determinants of

Synthetics Pigments

M J Scotter, Central Science Laboratory, Sand Hutton,

York, UK

Introduction

0001This article will review the use of synthetic coloring

materials in foodstuffs, structural classification,

chemical stability, interactions with other food

additives, usage in foods, and their qualitative and

quantitative analysis.

Classification

0002Synthetic food colors classification can be simplified

by grouping into the following chemical classes:

1556 COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments

.0003 Azo (monoazo, disazo, and trisazo)

0004 Azo-pyrazolone

0005 Triarylmethane

0006 Xanthene

0007 Quinoline

0008 Indigoid

Table 1 lists the food colors currently permitted in the

UK, European Union (EU), and USA and gives their

structural classes.

Azo Food Colors

0009 Azo dyes contain one or more chromophoric azo

groups which are usually associated with aromatic

systems containing salt-forming substituents gener-

ally in the meta or para position to the azo group.

Azo dyes span a wide range of colors: one example is

sunset yellow FCF (Figure 1a).

Azo-Pyrazolone Colors

0010 Azo dyes which also contain a pyrazolone group exist

essentially as keto-hydrazine tautomeric systems. A

well-known example of an azo-pyrazolone dye is

tartrazine (Figure 1b).

Triarylmethane Food Colors

0011Triarylmethane colors are characterized by the pres-

ence of a chromophoric system containing a central

carbon atom attached to three aromatic moeties with

amino, substituted amino and hydroxyl groups sub-

stituted in the para position, which act as auxo-

chromes. One example of a triarylmethane food dye

is green S (Figure 1c).

Xanthene Food Colors

0012Xanthene dyes are characterized by a chromophoric

system comprising essentially a dibenzo-1,4-pyran

heterocyclic ring system with amino or hydroxyl

groups in the meta position with respect to the

oxygen bridge. Erythrosine is the only example of a

xanthene dye currently permitted for food use in the

EU and the USA (Figure 1d).

Quinoline Food Colors

0013Quinoline yellow (Figure 1e) is the only example of

a quinoline dye currently permitted for food use in

the EU. The chromophoric system is based on the

2-(2-quinolyl)-1,3-indandione (or quinophthalone)

heterocyclic ring system.

Table 1 Synthetic food colors currently permitted in the European Union and USA

Name E number

FD&C classification

Colorindexno.

Structure type Color shade

Tartrazine E 102 Yellow no. 5 19140 Azopyrazolone Yellow

Quinoline yellow E 104 None 47005 Quinoline Greenish yellow

Sunset yellow FCF E 110 Yellow no. 6 15985 Monoazo Orange yellow

Carmoisine E 122 None 14720 Monoazo Bluish red

Amaranth E 123 None 16185 Monoazo Red

Ponceau 4R E 124 None 16255 Monoazo Orange red

Erythrosine E 127 Red no. 3 45430 Xanthene Bluish pink

Red 2G E 128 None 18050 Monoazo Bluish red

Allura red AC E 129 Red no. 40 16035 Monoazo Yellowish red

Patent blue V E 131 None 42051 Triarylmethane Violet blue

Indigo carmine E 132 Blue no. 2 73015 Indigoid Deep blue

Brilliant blue FCF E 133 Blue no. 1 42090 Triarylmethane Greenish blue

Green S E 142 None 44090 Triarylmethane Bluish green

Black PN E 151 None 28440 Disazo Bluish black

Brown FK E 154 None Azo

Orange brown

Brown HT E 155 None 20285 Disazo Dark brown

Lithol rubine BK

E 180 None 15850 Monoazo Bluish red

Citrus red

Red no. 2 12156 Monoazo Scarlet red

Fast green FCF Green no. 3 42053 Triarylmethane Bluish green

Orange B

Orange B 19235 Azopyrazolone Orange

As given in European parliament and Council Directive 94/326/EC on colors for use in foodstuffs.

As published by the US Food and Drug Administration.

As published by The Colour Index, Society of Colourists and Dyers, Bradford, UK.

Mixture of six main components; only used for coloring kippers.

For coloring of cheese rind only.

For coloring of orange skins only.

For coloring of casings or surfaces of frankfurters and sausages only.

Note: Yellow 2G is no longer permitted in the European Union.

COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments 1557

Indigoid Food Colors

0014 The only example of an indigoid dye currently per-

mitted for food use in the EU and the USA is indigo

carmine (Figure 1f), a blue-violet sulfonated analog

of indigo, a naturally occurring dye which exists

as a resonance equilibrium between two hybrid

structures.

Pigments and Lakes

0015Pigments are dyestuffs which are generally insoluble

in aqueous and organic solvents and have to be dis-

persed into foodstuffs to effect coloration. Precipita-

tion of water-soluble dyes on to an inert substrate

such as alumina forms water-insoluble pigments

known as lakes:

NaO

−

(a) (b)

(e)

(f)

N(CH

)

NaO

(NaO

NH HN

(CH

)

fig0001 Figure 1 (a) Chemical structure of azo dye sunset yellow FCF. (b) Chemical structure of azo-pyrazolone dye tartrazine. (c) Chemical

structure of triarylmethane dye green S. (d) Chemical structure of xanthene dye erythrosine. (e) Chemical structure of quinoline dye

quinoline yellow, showing the two main coloring components. (f) Chemical structure of indigoid dye indigo carmine, showing the two

main coloring components. Reproduced from Colours: Properties and Determination of Synthetic Pigments, Encyclopaedia of Food

Science, Food Technology and Nutrition, Macrae R, Robinson RK and Sadler MJ (eds) 1993, Academic Press.

1558 COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments

3(Dyestuff-SO



) þ Al

3þ

! (Dyestuff-SO



)

3þ

precipitated and extended on to Al

.3H

0016 No synthetic liposoluble dyestuffs are permitted in

the EU for the coloring of food. Synthetic nature-

identical dyestuffs are generally not considered as

synthetic food dyes.

Chemistry and Stability

0017 All of the synthetic coloring materials permitted for

food use in the EU and USA (except lithol rubine BK

and citrus red, which have restricted use), apart from

the lake colors, are soluble in water to a greater or

lesser extent and insoluble in fats. The degree of

water-solubility is determined by the number and

relative position of salt-forming groups present in

the dye molecule. The most common of these is the

sulfonic acid group SO

H and the less common

carboxylic acid group CO

H, which form water-

soluble anionic dyes. Cationic dyes contain basic

groups such as amino NH

or substituted amino

NH.CH

, N(CH

)

0018 Most food dyes are soluble in certain nonaqueous

hydrophilic solvents such as glycerine, propylene

glycol, and sorbitol, and this allows the preparation

of solutions and pastes for use in certain food prod-

ucts. The triarylmethanes and erythrosine are appre-

ciably soluble in the lower alcohols ethanol and

isopropyl alcohol. Turbidity or precipitation of color

may be experienced upon interaction with hard

water.

0019 Coloring materials exhibit excellent stability when

stored under cool, dry, and dark conditions. Many

factors can and do contribute to colorant stability,

such as heat, light, pH, redox systems, other food

ingredients (especially preservatives), and trace

metals.

Photodegradation

0020 Light is capable of inducing photochemical changes

in all dyestuffs, eventually leading to total decoloriza-

tion. Resistance to photochemical degradation is

termed light fastness. Heat, other various agents,

and food ingredients are known to accelerate the

photodegradation of dyestuffs, whereas others prove

to have a stabilizing effect. True azo dyes can undergo

three principal types of photochemical reaction; cis-

trans photoisomerism, photoreduction, and photo-

oxidation.

Thermal Degradation

0021 Heat can cause losses of color during food processing

and cooking. Coloring materials are added to prod-

ucts at the latter stages of and at the lowest possible

temperatures during food processing when further

heating is unlikely to take place. For all dyes, process-

ing at very high temperatures will lead to an

inevitable loss of color or change in shade due to

carbonization.

Acids, Alkalis, and Redox Systems

0022Not all colors can be used over all pH values and

some coloring materials, such as erythrosine, may

precipitate from solution at acid pH, whereas others

such as indigo carmine will fade rapidly. Color lakes

often exhibit amphoteric properties, with both acids

and alkalis tending to solubilize the inorganic sub-

strate and thus releasing the free colorant (i.e., color

‘bleed’).

0023The majority of permitted food colors exhibit

instability when used in combination with oxidizing

and reducing agents. Since color depends on the

existence of a conjugated unsaturated system within

the dye molecule, any substance which modifies

this system (e.g., oxidizing or reducing agents such

as hydrogen, sugars, acids, and salts) will affect

the color.

Metals

0024All dyes, those with the azo group in particular, will

exhibit accelerated fading under both acid and alka-

line conditions in the presence of metals including

zinc, tin, aluminum, iron, and copper, especially at

higher temperatures. This is mostly due to the

reducing effect of liberated hydrogen. Dyes will

often react with the metal in food cans at a rate

proportional to their concentration.

Interaction with Other Food Additives

Preservatives

0025Canned products containing added color may de-

grade in the presence of tartaric and citric acids,

which may react with the metal of the container to

liberate hydrogen. The stability of nine red colors in

comminuted meat products in the absence and pres-

ence of nitrite has shown that most of the dyes are

destroyed to some extent but with nitrite more of the

color survives. Subsidiary dye components and color-

less fluorescent products are formed as a result of heat

processing and in certain cases additional products

are observed in the presence of nitrite. Nitrite can also

cause rapid detinning to produce Sn

2þ

, a strong redu-

cing agent. Sulfur dioxide is known to cause rapid

decolorization of dye solutions. The interaction be-

tween sulfite and carmoisine results in the formation

of a hydrazo compound via hydrolysis.

COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments 1559

Ascorbic Acid

0026 Azo dyes are known to degrade under accelerated

conditions in the presence of ascorbic acid at pH 7

but are more resistant to degradation at pH 3. How-

ever, ascorbic acid at a concentration of 50 mg l

1

can

affect all synthetic coloring materials at pH 3 after 3

days’ storage at 20



Sugars

0027 Reducing sugars such as glucose and fructose can

reduce azo dyes in aqueous solution. Amaranth in

particular may degrade when incorporated into redu-

cing sugar-containing foods which are baked. The

presence of baking soda can promote the degradation

of the dye markedly in the presence of glucose.

Model Food Systems

0028 During accelerated storage in model soft drink

systems, tartrazine degrades very little. Amaranth

and sunset yellow FCF are also stable to most addi-

tives, with the exception of ascorbic acid and sodium

metabisulfite. In the latter case the degradation prod-

ucts appear to be higher-sulfonated analogs of the

parent dyes. Prolonged storage under these condi-

tions can therefore cause irreversible degradation

leading to colorless products. The formation of spe-

cific amines from the degradation of amaranth has

been used to estimate the amounts of dye added to

soft drinks. Red dyes have been shown to degrade

both before and after storage in fish paste products.

Ponceau 4R may be reduced to a yellow by hydrogen

sulfide or sulfur compounds liberated during process-

ing and storage of certain foodstuffs, and erythrosine

may lose iodine to produce fluorescein when incorp-

orated in canned cherries and stored in unlacquered

cans.

Color Usage in Processed Foods

0029 Certain food processes have been associated with the

use of food coloring matter for some time, principally

to:

0030 reinforce colors already present in foods to meet

consumer expectations

0031 insure uniformity of colour in batch productions

0032 restore the original appearance of certain foods

when color has been, or will be, diminished during

processing or storage

0033 give color to otherwise colorless foods such as

candies (sweets), instant desserts, and ice lollies

Table 2 gives a brief summary of color usage in foods

in the UK in 1987.

Dye Purity

0034Most of the dyes used for coloring foods comprise

several colored components as well as the main dye.

These are collectively known as subsidiary colors.

The manufacture of a dye from its starting materials

usually involves a number of synthetic stages and

transformations such as reduction, amination, sulfon-

ation, diazotization, condensation, and oxidation.

The side-reaction products and the precursors of

the dyes themselves are collectively known as ‘inter-

mediates’ and in food dyes these are often sulfonated

compounds. Table 3 lists those most commonly

found in food colors. Coloring material specifica-

tions in general therefore contain criteria for limita-

tions on subsidiary dyes and intermediates as well as

certain unsulfonated or free aromatic amines. Separ-

ate criteria are prescribed for inorganic impurities

such as transition metals, heavy metals, and certain

salts.

Inorganic Impurities

0035The most commonly found inorganic impurities in

food colors are sodium chloride and sodium sulfate.

Small amounts of phosphate, acetate, carbonate,

and iodide may also be present. The criteria for purity

with respect to inorganic matter are somewhat

different for lake colors.

Organic Impurities

0036The most common organic impurities present in

synthetic coloring materials are small amounts of

reaction intermediates. There may be various unsul-

fonated aromatic compounds as well as the sulfon-

ated analogs present in the finished colorants owing

to impurities in the starting materials. Triarylmethane

dyes are prepared by condensation reactions during

which an uncolored leuco base is formed as an inter-

mediate. The leuco base is then oxidized to the fully

conjugated colored dyestuff using oxidizing agents

such as lead dioxide, manganese dioxide, or dichro-

mate, which might then be present as low-level

inorganic contaminants in the finished dye.

Analysis of Synthetic Food Colors

0037The major components of synthetic water-soluble

food color formulations are active dye (including

subsidiary dyes), inorganic salts, and moisture.

Other constituents may be permitted diluents or ex-

tenders which may be added for standardization pur-

poses or to facilitate the incorporation of the colorant

for certain applications.

1560 COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments

tbl0002 Table 2 Guide to synthetic food coloring usage in the UK

Commodity class Food types Stability

requirements

Additional comments Permitted colors

Typical levels

of application

(mg kg

1

or mg l

1

)

Soft drinks and other

nonalcoholic

beverages

Ready-to-drink cordials,

vending machine

concentrates, instant

teas

LF, AC, PR,

FL, TM

Must not accelerate

corrosion of metal

containers

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT

10–100

Proportionally

higher in

concentrates

Alcoholic

beverages

Beers, ciders, fortified

and aromatized wines,

spirituous beverages

SD Limited use TZ, SY, AM,

P4R, CA, BB,

GS, AR, BV,

IC, QY, BHT,

BPN

Up to 200

Confectionery Boiled sweets, toffees,

caramels, gums, jellies,

pastilles, licorice,

chewing gum

TM, SD, FL,

Added as late as

possible during

production; colors

often bright/intense

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

50–300

Decorations and

coatings

Fine bakery wares Biscuits, wafers, cakes,

baking ingredients

TM Raising agents may

be present

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

Up to 200

Soups TM, AC Requires consistent

staining of product

relative to carrying

liquors

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC, QY,

BHT, BPN

Up to 50

Meat products Breakfast sausage TM, PR Must show stability

in and affinity

for protein

AR, R2G 25, 20

Burger meat AR, R2G -, 20

Luncheon meat AR 25, -

Meat and fish

analogs

Based on vegetable

proteins

TM, PR Must show stability

in and affinity for

protein, Lakes often

used

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

Up to 100

Fish and fish

products; shellfish

Smoked fish TM, PR Must show stability

in brine and affinity

for protein; surface

coloring often used

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN;

AM only in

fish roe

100

Salmon substitutes 500

Surimi 500

Fish roe 300

Fish paste and

crustacean paste

100

Precooked

crustaceans

250

Kippers BFK 20

Cheese Flavored processed

cheese

AC, PR Color must not

migrate from

rind/casing into

cheese

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

(LBK cheese

rind only)

100

Edible cheese rind QS

Edible cheese casing QS

Special dietary foods Solid food supplements Various Most have special

requirements

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

Up to 300

Liquid food

supplements

Up to 100

Complete formulae Up to 50

Preserves Jam, jelly, and

marmalade

TM, AC Must be stable to high

temperatures found

in jam-making and

migration

GS, P4R, SY, QY Up to 100

Preserves of red fruits

Candied fruits

Desserts, including

flavored milk

products

Blancmanges, custards,

mousses, dry mixes,

sauces

TM, LF, GE Lakes often used but

must not show

speckiness in product

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

Up to 150

Edible ices, icecream

Continued

COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments 1561

Dye Content

0038 Azo, triarylmethane, and indigoid dyes are readily

reducible by tin (II) chloride, which forms the basis

of the titrimetric assay method for dye content.

Erythrosine (xanthene type) and quinoline yellow

(quinophthalone type) are not reducible and have to

be assayed by other means. However, erythrosine is

the only commonly used food dye which is insoluble

in dilute acid and which can consequently be assayed

gravimetrically.

0039 Simple spectrophotometric methods are commonly

used for the assay of food dyes. Measurement in the

ultraviolet/visible range on instruments with scanning

and recording facilities is common. The use of high-

performance liquid chromatography (HPLC) for the

determination of dyestuff purity is also widespread.

Inorganic Salts

0040 The classical method for the determination of chlor-

ide is by the precipitation of chloride as its silver salt.

Sulfate may also be determined gravimetrically as the

barium salt. Electrometric procedures may also be

used for specific ion determination. Chloride, sulfate,

and other ionic species may also be determined

simultaneously using ion chromatography.

Moisture

0041Standard procedures for moisture determination are

loss on drying, or by nonaqueous titrimetric tech-

niques such as the Karl–Fischer procedure.

Lakes

0042Alumina is one of the major components of food lakes

but is rarely determined. Food lakes are generally

soluble in hot dilute ammonia solution and all alu-

mina lakes, with the exception of erythrosine,

dissolve readily in hot dilute hydrochloric acid. The

resultant solutions give the identifiable reactions of

aluminum with base and with alizarin. Qualitative

identification of the parent dyes and quantitative an-

alysis of the major and minor components of lakes

can be carried out using similarly prescribed proced-

ures as for the water-soluble analoges. Certain lake

colors such as erythrosine may prove difficult to ana-

lyze and may require special procedures for analysis.

Commodity class Food types Stability requirements

Additional

comments

Permitted colors

Typical levels

of application

(mg kg

1

or mg l

1

)

Snack foods: dry,

savoury potato,

cereal- or

starch-based

Extruded or expanded

savoury products

LF, TM Products often

surface-treated

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

Up to 200

Other savoury

products and nuts

Up to 100

Sauces, seasonings Pickles, relishes,

chutney, curry

powder, tandoori

TM, AC, LF Most have special

requirements; dye

mixtures often used

TZ, SY, P4R,

CA, BB, GS,

AR, BV, IC,

QY, BHT, BPN

Up to 500

Mustard Up to 300

Other Cocktail and candied

cherries in syrup

and cocktails

Cherry color must

not leach

ERY 150–200

Processed mushy and

garden peas (canned)

TM, AC Requires

consistent

coloring

TZ, GS, BB Up to 100

Based on Ministry of Agriculture, Fisheres, and Food (1987) UK Survey of Colour Usage in Food, Food Surveillance paper no. 19. London: HMSO; and

European parliament and Council Directive 94/326/EC on colors for use in foodstuffs.

LF, light-fastness; AC, acids; PR, preservatives; FL, flavorings; SD, sulfur dioxide; GE, gelling agents and emulsifiers; TM, temperature.

TZ, tartrazine; SY, sunset yellow FCF; P4R, ponceau 4R; AM, amaranth; CA, carmoisine; BB, brilliant blue FCF; GS, green S; BHT, brown HT; IC, indigo

carmine; BPN, black PN; ERY, erythrosine; R2G, red 2G; BFK, brown FK; BV, patent blue V; QY, quinoline yellow; AR, allura red; LBK, lithorubine K; QS,

quantum satis (no maximum level specified).

Commodity class Food types Stability

requirements

Additional comments Permitted colors Typical levels

of application

(mg kg

1

or mg l

1

)

Table 2 Continued

1562 COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments

Minor Components

0043 Water-insoluble matter is calculated on the basis of

100% dye content and is usually determined by gravi-

metric procedures. Atomic absorption spectropho-

tometry has largely superseded the use of wet

methods for trace heavy metals, mainly because of

the speed with which analyses can be carried out and

the high levels of accuracy and precision which can be

attained. Other emergent techniques which have been

used with limited application because of their high

costs are inductively coupled plasma spectroscopy

(ICPS), X-ray fluorescence (XRF, for mercury), and

neutron activation analysis (NAA).

Organic Impurities

0044Ether-extractable matter content is usually deter-

mined by Soxhlet extraction and is applicable to all

dyes except erythrosine.

0045Primary aromatic amines are usually determined

by diazotization and coupling (to N-1-naphthyl-

ethylenediamine, NED) of an appropriate extract,

followed by spectrophotometric measurement or

tbl0003 Table 3 Intermediate compounds commonly found in synthetic food colors

Intermediatename Trivialname Occurrence

Aminobenzene Aniline R2G

1-Aminobenzenesulfonic acid Sulfanilic acid BPN,SY,TZ,Y

2G,BFK

4-Aminonaphthalene-1-sulfonic acid Naphthionic acid AM,CA,P4R,B

6-hydroxynaphthalene-2-sulfonic acid Schaeffers acid AM,P4R,SY,A

R,GS

3-hydroxynaphthalene-2,7-disulfonic acid R-Acid AM,GS,P4R,S

7-Hydroxynaphthalene-1,3-disulfonic acid G-acid AM,P4R

7-Hydroxynaphthalene-1,3,6-trisulfonic acid AM,P4R

4-Acetamido-5-hydroxynaphthalene-1,7-disulfonic acid Acetyl K-Acid BPN

8-Aminonaphthalene-2-sulfonic acid 1,7 Cleves acid BPN

4-Amino-5-hydroxynaphthalene-1,7-disulfonic acid K-Acid BPN

4-Hydroxynathalene-1-sulfonic acid N & W Acid CA

5-Amino-4-hydroxynaphthalene-2,7-disulfonic acid H-Acid R2G

5-Acetamido-4-hydroxynaphthalene-2,7-disulfonic acid Acetyl H-Acid R2G

4,43-Diazoaminodi(benzenesulfonic acid) Triazene SY,TZ,Y2G

6,63-Oxydi(naphthalene-2-sulfonic acid) DONS SY

Tetrahydrosuccinic acid Dioxytartaric acid TZ

4-Hydrazinobenzenesulfonic acid TZ

5-Oxo-1-(4-sulfophenyl)-2-pyrazoline-3-carboxyllic acid SPCZ TZ

2,5-Dichloro-4-(3-methyl-5-oxo-2-pyrazolin-1-yl)

benzenesulfonic acid

CSPMZ Y2G

Fluorescein ERY

2,4,6-Triiodoresorcinol ERY

2-(2,4-dihydroxy-3,5-diiodobenzoyl)benzoic acid ERY

1-H-Indole-2,3-dione (and analogous sulfonic acids) Isatin IC

5-Sulfoanthranilic acid IC

1-H-Indole-2,3-dioxo-1-H-indole-5-sulfonic acid Monosulfonated indigo IC

2-, 3-, and 4-Formylbenzenesulfonic acids BB

N-Ethyl-N-(3-sulfobenzyl)sulfanilic acid ESBSA BB

m-Phenylenediamine BFK

4-methyl-m-phenylenediamine BFK

4,43-Bis(dimethylamino)benzhydrol alcohol GS

4,43-Bis(dimethylamino)benzophenone GS

N,N3-Diethylaniline BV

m-Hydroxybenzaldehyde BV

2,4-Dihydroxybenzylalcohol BHT

5-Amino-4-hydroxy-2-toluenesulfonic acid AR

p-Hydroxybenzaldehyde-o-sulfonic acid FG

a-(N-Ethylanilino)-m-toluenesulfonic acid FG

BPN, Black, PN; SY, Sunset yellow, FCF; TZ, Tartrazine; Y2G, Yellow 2G; BFK, Brown FK; AM, Amaranth; CA, Carmoisine; P4R, Ponceau 4R; BHT, Brown

HT; R2G, red 2G; Ery, Erythrosine; IC, Indigo carmine; BB, brilliant blue FCF; GS, Green S; BV, patent blue V; AR, Allura red AC; FG, Fast green FCF.

COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments 1563

HPLC. HPLC has superseded classical column chro-

matographic procedures as the most widely used

technique for the separation and quantitation of inter-

mediate species. Both gas and thin-layer chromatog-

raphy have also been used for the determination of

certain intermediate compounds. The leuco base con-

tent of triarylmethane dyes is determined by carrying

out further oxidation of the parent dyestuff and

measuring the subsequent increase in absorbance.

Analysis of Foodstuffs for Synthetic

Coloring Materials

0046 Essentially food colors must first be extracted from

the food matrix, purified to remove potentially

interfering coextractives, and concentrated prior to

identification and quantitation. Some form of sample

pretreatment is often required, such as defatting of

meat products or dilution of sugars and gums in

confectionery products, before the extraction can

proceed.

Leaching

0047 Leaching may be used to remove colorants from the

surface of foodstuffs such as sausages, and also from

food-packaging materials. In the simplest applica-

tion, the sample is soaked in an appropriate (usually

alkaline) solvent which is then filtered or centrifuged

to clarify the colorant solution. Further clean-up is

performed as necessary. Newer techniques such as

supercritical fluid extraction (SFE) may prove to be

useful for the extraction of colorants, intermediates,

and interaction products from foods.

Solvent-Solvent Extraction and Ion-Pair

Techniques

0048 These are widely used and effective methods of color-

ant isolation. Simple immiscible solvent pairs may be

used where one solvent acts as a carrier for a dye-

complexing reagent or soluble ion-exchange resin.

The higher alcohols, particularly 1- and 2-butanol,

are the most useful solvents for this technique.

Amberlite LA-2, a liquid anion-exchange resin dis-

solved in butanol (or hexane), has been widely used

as a dye-extraction medium for foodstuffs.

0049 Quaternary ammonium compounds such as cetyl-

cyclohexyl-dimethylammonium bromide (biocidan)

and cetyltrimethyl-ammonium bromide (cetrimide)

have been used for the extraction of synthetic dyes

from food. More recently, reagents such as tetra-

n-alkylammonium halides and cetylpyridinium chlor-

ide have also been employed for the rapid extraction

of anionic dyes, as hydrophobic ion-pair complexes,

from food using organic solvents.

Enzymatic Digestion

0050Pretreatment of a food sample by enzymatic digestion

may be used prior to extraction of the colorants in

order to release those colorants which may be highly

bound or associated with the food matrix. Enzyme–

substrate combinations that may be selected include

papain (for protein digestion), lipase (lipids), phos-

pholipase (phospholipid), amyloglucosidase (starch),

pectinase (pectin), and cellulase (cellulose). Opti-

mization of the pH and temperature conditions is

necessary in each case.

Adsorption Techniques

0051Adsorption, generally referred to as solid-phase ex-

traction (SPE), techniques have been developed for

the isolation of food colorants utilizing a variety of

adsorption materials such as wool, powdered leather,

cellulose, alumina, and polyamide powder. More

recently, semimicro adsorption cartridges containing

reverse-phase bonded silica materials have found

widespread use. Adsorption is achieved by either

adding adsorbent directly to a pH-adjusted sample

solution or by passage of the sample solution through

a column packed with adsorbent. The absorbent is

freed of other sample matrix components by washing

with appropriate solvents and the colorants select-

ively desorbed using a different solvent, similar to

those used in HPLC.

Dialysis Techniques

0052Dialysis has had limited application in the extraction

and concentration of synthetic dyes from foods but

has been used for the isolation and concentration of

certain dyes from soft drinks and sugar-rich foods

such as jellies and boiled sweets. These techniques

are usually automated and employ SPE cartridges

followed by HPLC.

Qualitative and Quantitative Analysis of

Food Extracts

Spectrophotometry

0053Numerous techniques are available for the spectro-

photometric analysis of colorants: measurements at

ultraviolet and visible wavelengths are the easiest to

perform. Beer’s law can simply be applied to extracts

containing single colors, whereas extracts containing

two or more colors can be problematic. If the iden-

tities of the coloring components are known, their

concentrations can be determined providing there is

no interaction between them. The distinguishing fea-

tures of the spectra obtained for single colors may be

significantly affected by careful adjustment of the pH

1564 COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments

of the solution with acid or alkali, characterized by

shifts in absorption wavelength maxima and inten-

sities. Simultaneous spectrophotometric determin-

ation of mixtures of food colorants without prior

chemical separation has been applied to mixtures of

up to five dyes. The data obtained are processed by

chemometric approaches which utilize normal ab-

sorbance spectra as well as first- and second-order

derivative spectra.

0054 Other spectroscopic techniques, such as infrared,

Raman (e.g., surface-enhanced scattering tech-

niques), and nuclear magnetic resonance, have been

used for the analysis of food colors but do not lend

themselves to routine application.

Mass Spectrometry

0055 Because of their inherent lack of volatility, direct

analysis of sulfonated azo (and other) food dyes by

mass spectrometry (MS) is very difficult. Several re-

action schemes may be used to obtain various volatile

neutral derivatives to facilitate analysis. Alternative

ionization techniques other than electron impact (EI)

and chemical ionization (CI) may be used for the

analysis of food dyes but so far have had limited

application. The most useful of these are fast-atom

bombardment (FAB), field desorption (FD), and

secondary-ion MS (SIMS). Liquid chromatography–

mass spectroscopy techniques are beginning to find

use as ‘benchtop’ instruments become more wide-

spread. Atmospheric pressure chemical ionization

(APCI) in positive and negative ion modes as well as

pneumatically assisted electrospray have been used to

determine mono- and disulfonated azo dyes.

Polarography

0056 Differential pulsed polarography and differential

pulse adsorptive stripping voltammetry may be used

to estimate dye concentrations in food matrices. The

addition of gelatin has been found to be advantageous

in the partial identification and determination of food

colors due to its pronounced affects on measured

peak currents.

Electrophoresis

0057 Of the various electrophoretic techniques available,

paper electrophoresis has been the most widely used

for the analysis of food colors, utilizing a range of

different buffer systems and applied potentials. Cel-

lulose acetate and polyacrylamide gel have been used

for the electrophoretic separation of azo and triaryl-

methane dyes.

0058 Capillary zone electrophoresis (CZE) is an analyt-

ical technique that has proven to be applicable to the

analysis of food dyes, especially when coupled to

photodiode array detectors. The separation of syn-

thetic dyes is influenced by buffer composition, pH,

and additives such as cyclodextrins. The related tech-

nique micellular electrokinetic capillary chromatog-

raphy (MEKC) has also found application in the

analysis of synthetic food dyes. Mixtures of food

dyes may be separated and determined simultan-

eously. Buffered mobile phases modified with aceto-

nitrile or methanol and containing other additives

are often used. Both techniques may be automated

readily.

Gas Chromatography

0059Gas chromatography cannot be used for the direct

analysis of food dyes owing to their inherent lack of

volatility. It is however a useful technique for the

analysis of volatile derivatives and certain inter-

mediate compounds.

Paper Chromatography

0060Paper chromatographic techniques have been widely

used in the past for the identification of food colors

but have nowadays been largely superseded by thin-

layer, column, and HPLC techniques. Paper chroma-

tography maintains popularity in some laboratories

because of its relatively low cost and ease of use, and

many suitable solvent systems are available.

Thin-Layer Chromatography

0061The use of thin-layer chromatographic systems for the

separation of food dyes is fairly widespread, but is

gradually being superseded by HPLC. Silica gel

is the most commonly reported adsorbent used,

though alumina, microcrystalline cellulose, and high-

performance reverse-phase bonded silicas have

widespread use. High-performance thin-layer chro-

matography with densitometric detection has been

used for the determination of dyes in alcoholic and

nonalcoholic beverages.

High-Performance Liquid Chromatography

0062HPLC has become the major analytical technique for

the determination of synthetic coloring materials in

foodstuffs. The most widely used separation modes

are ion-exchange and reverse-phase. Spectrophoto-

metric detection is applied in the visible wavelength

range for dyes and subsidiary colors or in the ultra-

violet range for intermediates and other organic

impurities.

Ion-Exchange HPLC

0063Dyes, subsidiary colors, intermediates, and impurities

have all been characterized using ion exchange,

COLORANTS (COLOURANTS)/Properties and Determinants of Synthetics Pigments 1565