Categories of natural pigments, Natural food colours, Detection of natural colours, Analysis of natural colours

1. PIGMENT ANALYSIS
Dr Gana Manjusha K
Associate Professor
Vignan Institute of Pharmaceutical Technology

2. Definition and Importance
• Pigments are chemical compounds that absorb light
in the wavelength range of the visible region.
Produced color is due to a molecule-specific
structure (chromophore).
• The natural color of foods of plant origin is due
primarily to four groups of pigments: the green
chlorophylls, the yellow-orange-red carotenoids, the
red-blue-purple anthocyanins, and the red
betacyanin.
• A few animal-derived foods are colored by
carotenoids. These pigments are also incorporated
into food products by direct addition or indirectly
through animals’ feed.

3. Importance of Analysis
• Although earlier investigations were motivated by the
color, recent studies have been stimulated by potential
health effects.
• These possible health benefits, along with consumers’
concern about safety, have led to efforts to replace
artificial food dyes with natural colorants. This is not
an easy task, however.
• Natural colorants are usually less stable, more costly,
and not as easily utilized as synthetic dyes, besides
having weaker tinctoral strength, interaction with food
components, and limited range of hues.

4. Green Chlorophylls
Yellow-orange-red
Carotenoids
Red-blue-purple
Anthocyanins
Red Betacyanin
• Four groups of Natural pigments

5. Anthocyanins
• Anthocyanins are water-soluble pigments
belonging to the family of compounds
called flavonoids.
• They are glycosides or acylglycosides of six
commonly found aglycone anthocyanidins:
pelargonidin, cyanidin, delphinidin,
peonidin, petunidin, and malvidin (Fig. 1).
• Cyanidin is the most commonly occurring
anthocyanidin in nature.

7. Chemical Structures, Properties, and Occurrence
• Anthocyanidins are flavylium (2-phenylbenzopyrylium)
structures with varying hydroxyl and methoxyl
substituents.
• They consist of two aromatic rings (rings A and B)
linked by a three carbon heterocyclic ring (ring C) that
contains oxygen.
• Eight conjugated double bonds carrying a positive
charge on the heterocyclic oxygen constitutes the
chromophore responsible for the intense color of
anthocyanins.
• The color of anthocyanin-rich fruits and vegetables vary
from vivid red as in strawberry, purple as in eggplant,
and dark blue as in blueberry.

8. • In aqueous medium such as foods, anthocyanins
undergo reversible structural transformations with
pH (Scheme 1), with concomitant change in color.
• The red favylium cation predominates at pH below
2. At pH 3–6, rapid hydration of the flavylium
cation occurs at C-2 to form the colorless carbinol
pseudobase.
• The carbinol by ring-opening tautomerization gives
rise to the (Z)-chalcone, which can isomerize to the
(E)-chalcone.
• At slightly acidic to neutral conditions,
deprotonation of the flavylium cation generates the
blue quinoidal base.

10. • Anthocyanins are found in fruits (especially
berries), vegetables, nuts, grains, roots, and
flowers. Major sources are purple grapes,
cherry, plum, raspberry, strawberry,
blackberry, blueberry, black currant, cranberry,
chokeberry, red cabbage and red wine.
• The anthocyanin composition of foods is
affected by several factors, such as
cultivar/variety, maturity, cultivation practices,
growing area or season/climate and storage
conditions.

11. Betacyanins
• Betalains are water-soluble nitrogen-
containing pigments, consisting of the red to
red-violet betacyanins and yellow-orange
betaxanthins.

12. Chemical Structures, Properties, and
Occurrence
• They are immonium conjugates of betalamic acid with
cyclo-dopa[cyclo-3-(3,4-dihydroxyphenylalanine)] and
amino compounds, respectively.
• The bathochromic shift of 50–70 nm of betacyanins
compared to betaxanthins is due to the aromatic ring of
cyclo-dopa.
• Glycosylation of betanidin results in a hypsochromic
shift of the resulting betacyanin with glucose attached at
C-6 being less effective than that linked to C-5.
• Esterification with aliphatic acyl moieties had little
impact on the maximum absorption of betacyanins.

13. • For a long time, beetroot was considered the sole
source of betacyanin for use as food colorant,
betanin (betanidin 5-O-β-glucoside) being the most
abundant pigment.
• Recent years have seen an increased search for other
sources, such as Ullucus tuberosus, one of the most
widely grown and economically important root crops
in the Andean region of South America; Basella
rubra, commonly known as Malabar spinach, a leafy
vegetable that accumulates pigments in its fruits;
cactus pear (Opuntia ficus-indica and O. stricta);
red-purple pitaya Hylocereus polyrhizus; and
Amaranthus species.

15. Carotenoids
• Chemical Structures, Properties, and Occurrence
• The lipophilic carotenoids are generally C40
tetraterpenes/tetraterpenoids formed from eight C5
isoprenoid units joined head to tail, except at the center
where a tail to tail linkage reverses the order.
• The most distinctive structural feature is a centrally
located, conjugated double bond system.
• The basic linear and symmetrical skeleton is modified in
many ways, including cyclization, hydrogenation,
dehydrogenation, introduction of oxygen containing
groups, migration of the double bonds, rearrangement,
chain shortening or extension, or combinations thereof,
resulting in a wide array of structures.

16. • Carotenoids may be acyclic (e.g., lycopene, ζ-
carotene) or may have a six membered ring at
one (e.g., γ-carotene, δ-carotene) or both ends
(e.g., β-carotene, α-carotene) of the molecule,
except capsanthin and capsorubin which have
five membered rings.
• Hydrocarbon carotenoids (e.g., β-carotene,
lycopene) are known as carotenes, and the
oxygenated derivatives are called xanthophylls.

17. • Carotenoids are not as widely distributed in
foods of animal origin and the composition
is simpler.
• Unable to carry out carotenogenesis,
animals are limited to absorbing dietary
carotenoids, which are accumulated
unchanged or slightly altered to form
carotenoids typical of animal species.

18. • Natural examples are bixin, the major
pigment of annatto, and crocetin, the main
coloring component of saffron.
• The carotenoids most commonly
encountered in foods are β-carotene, α-
carotene, β-cryptoxanthin, lycopene, lutein,
and zeaxanthin .
• Rich sources of β-carotene are some palm
fruits, some squash cultivars, green leafy
and non-leafy vegetables, carrot, orange-
fleshed sweet potato, cantaloupe, mango,
and apricot.

19. • β- Carotene is sometimes accompanied by α-carotene, as in
carrot, some varieties of squashes, pumpkins, and palm
fruits.
• β-Cryptoxanthin is the main carotenoid of orange-fleshed
fruits, such as papaya, tangerine, orange, loquat,
persimmon, and tree tomato.
• Lycopene-rich foods are tomato and tomato products,
pitanga, pink-fleshed guava, red-fleshed papaya, and
watermelon.
• The richest sources of lycopene, however, are the Asian gac
(Momordica cochinchinensis) fruit and the Spanish
sarsaparilla .
• Corn and corn products and some varieties of squash are
the major dietary sources of zeaxanthin/lutein. Leafy and
other green vegetables are the main sources of lutein.

21. Chlorophylls
Structures, Properties, and Occurrence
• Chlorophyll a and b are the typical green pigments of
higher plants, occurring in an approximate ratio of 3:1
in lettuce leaves and in five commonly consumed
Mediterranean leafy vegetables, but with a ratio of 1:2
to 1:4 in different varieties of the tree tomato fruit.
• Chlorophylls are porphyrins, which are macrocyclic
tetrapyrrole pigments in which the pyrrole rings are
joined by methyne bridges and the double bond system
forms a closed, conjugated loop.
• A centrally located magnesium atom is coordinated
with the nitrogen of the four pyrroles.

22. • Chlorophyll a has a methyl group and chlorophyll b has
a formyl group at C-3.
• Both have a vinyl and an ethyl group at the C-2 and C-4
position, respectively; a carbomethoxy group at the C-10
position of an isocyclic ring; and a phytol group
esterified to propionate at the C-7 position.
• Phytol is a 20-carbon monounsaturated isoprenoid
alcohol.
• Chloropyll a appears blue-green and chlorophyll b
yellow- green.
• The chlorophyll molecule has a hydrophilic part, the
macrocycle, and a hydrophobic segment, the phytol.
• The closed circuit of conjugated double bonds is the
chromophore that allows them to absorb light.

24. ANALYSIS
• β-carotene: It is commonly called C.I. natural yellow 26 and belongs
to the class of carotenoids. It contains all trans-β-carotene; no cis β-
carotene should be present. The general requirements of this compound
can be divided into two different types as follows:
• Spectrophotometric requirement: The absorption maxima in
cyclohexane in a 1 cm cell should be in the region of 456 to 484 µm.
No peaks at 330 to 355 µm should be observed as it represents a cis
peak.
• Colour reaction: β-carotene should have a purity of NLT 96%. The
other requirements are:
• Arsenic should be NMT 3 parts per million (ppm)
• Lead should be NMT 10 ppm
• Heavy metal content should be NMT 40 ppm
• Subsidiary colouring matter should be NMT 3% by mass
• Sulphated ash and total colouring matter should be NMT 0.1%

25. • Chlorophyll: Chlorophyll, also known as C. I. Natural
green 3, is a plant-based green pigment is used as a
colouring material for foods.
• The following tests confirm that chlorophyll fulfils the
requirements for being able to be used in food items:
• Identification test: A chlorophyll solution in ethanol should
produce blue colour with deep red fluorescence.
• Brown phase reaction: When 10% methanolic potassium
hydroxide is added to the solution of chlorophyll made
with green ether or petroleum ether, it should turn brown
and then back to green quickly. This test does not give a
positive result if the chlorophyll is treated with alkalis.

26. • Limits for metallic impurities in chlorophyll are:
• Arsenic should be NMT 3 ppm
• Lead should be NMT 10 ppm
• Copper should be NMT 30 ppm
• Zinc should be NMT 50 ppm
• Total combined phaeophytines and their magnesium
complexes should be NMT 10% by weight.
• The level of residual solvents like methanol, ethanol,
acetone, 2-propanol, and hexane should be NMT 50
mg/kg. The residual solvent content of
dichloromethane should be NMT 10%.

27. • Riboflavin: It is yellow to orange-yellow and
approved for the use in food products as a
colourant. It should fulfil the following
requirements if it has to be safely used in food:
• Identification test: 1% riboflavin solution in
water should be pale greenish yellow in
transmitted light; it should have deep yellowish
green fluorescence. This intense fluorescence
should disappear on the addition of mineral
acids or bases and sodium dithionite.

28. • Spectrophotometry: Maximum absorption
of an aqueous solution of riboflavin
should be in the region of 220 to 225, 266,
371, and 444 μm.
• Total minimum purity of riboflavin: 97%
• Maximum limit of metallic impurities:
– Arsenic should be NMT 5 ppm
– Lead should be NMT 20 ppm

29. Detection of Natural Colours
Caramel: Caramel is detected by Fiehe's
reaction. Extract the sample solution with 50 ml
ether and evaporate it in a porcelain dish. To
the residue add 3 drops of 1% solution of
resorcinol in HCl. The presence of caramel is
indicated by appearance of rose colour.

30. • Cochineal: Shake an amyl alcohol solution of
the material with dilute ammonia. A purple
colour is produced in the presence of cochineal.

31. • Turmeric (curcurnin): Evaporate an
alcoholic extract of the material almost to
dryness on the water bath with a piece of
filter paper. Moisten the dried paper with
afew drops of weak solution of boric acid to
which some drops of HCI have been added.
Dry the paper again. If turmeric is present,
the dry paper will be cherry red in colour
which changes to bluish green by a drop of
NaOH or NH4OH.

32. • Annatto: Shake the melted fat or oil with
2% NaOH solution and pour the aqueous
extract on moistened filter paper. The filter
paper will show a straw colour which will
remain with a gentle wash with water. Dry
the paper and add a drop of 40% stannous
chloride solution and dry carefully. If the
colour turns purple, the presence of annatto
is' confirmed.

33. • Chlorophyll: Extract the sample with ether
and treat the ether extract with 10% KOH in
methanol. Colour becomes brown, quickly
returning to green, confirms the presence of
chlorophyll.
• Betanin: Extract the aqueous suspension
with amyl alcohol. It remains in aqueous
phase. Dye it with a piece of tannin
mordanted cotton, a terracotta shade is
produced in presence of betanin.