PIGMENTS AND DYES, food analysis, analysis

KARNATAKA COLLEGE OF PHARMACY
1
TOPIC: PIGMENTS AND SYNTHETIC
DYES
PRESENTED TO:
DR.HARSHA K. TRIPATHY
PROFESSOR
DEPT. OF PHARMACEUTICAL
ANALYSIS
KARNATAKA COLLEGE OF
PHARMACY
BANGALORE
PRESENTED BY:
S.GOKULRAJ
M PHARM 1ST SEMESTER
DEPT.OF PHARMACEUTICAL ANALYSIS
BANGALORE
SUBJECT: FOOD ANALYSIS

INTRODUCTION OF PIGMENTS
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 A pigment is a natural colouring matter found in plant or animal cells or
tissues.
 Pigments derived from word pigmentum means coloring matter.
 Pigments are organic and inorganic material which are practically
insoluble in medium.
 Pigments are used almost 30000 years ago.
 The first synthetic pigment was prussion blue which was synthesized in
1704.

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 A food pigments either synthetic or natural, which imparts colours to the
food.
 The acceptance of a food depends to a large extent upon its attractive
colour.
 Naturally present in the cells and tissue of plants.
 These are the most important factors, which are evaluated by the consumers
while purchasing the foods.
 Pigments occurs in many forms consisting of liquids,powders,gels and
pastes.
 Pigments are used for coloring paint, ink, plastic, fabric, cosmetics, food
and other materials.

Characteristic of pigment
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 Good resistance to light, wetness and abrasion.
 Good chemical resistance.
 Power to mix freely.
• Chemical inertness.
• Excellent dispersion
 Highly durable, Highly heat stable, Solvent resistant.
 Pigments have acceptable brilliance, hardness and stability.
 Hard to process

Properties of Pigments
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 No auxochrome group.
 Applicable to all kinds of fibers or textile materials.
 Rubbing fastness is poor.
 Particle size: 0.2-0.4 μm
 Specific gravity:1.14-1.37
 Boiling points : 190-345°C
 Melting points: 110-175°C
 Insoluble in water & other solvents like white spirit, per chloroethylene,
trichloroethylene, CCI4 etc.
 Most of the pigments are azoic compounds. Besides that it may be inorganic
oxide, inorganic salt phthalocyanine metal compounds etc.
 Pigments are toxic. Some are oral toxic, some are dermal toxic, some causes eye
irritation etc.

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Selection of pigments:
 Refractive index
 Light fastness
 Bleeding characteristic
 Particle size and shape
Application:
 Pigments are used for coloring of leather, building materials, paper, floor
covering, ceramic glazes, paint, ink, plastic, fabric, cosmetics, food, and
other materials.
Types of pigments
1. Organic pigments
2. Inorganic pigments

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1. Organic pigments:
 Organic pigments are usually brighter, stronger, and more transparent than
inorganic pigments but are not as light resistant.
 Organic pigments are based on the carbon chain and carbon rings.
 They can also contain metallic elements that help stabilizing the properties
of the organic components.
 It will be present in nature.

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Characteristic of organic pigments:
 High colour strength
 Give bright shades
 High light fastness
 Good weather fastness
Types of organic pigments:
1. Azo pigment
2. Non azo pigments
 Polycyclic
 Dioxazine
 Anthraquinone
 Quinophthalone
 Triaryl Carbonium

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2.Inorganic pigment:
 Inorganic pigments use chemical formulations to get the desired product
properties for various applications.
 Compounds obtained from inorganic metallic compounds and salts such as
chromates, metallic oxides, sulphates etc. are used in inorganic pigments.
Characteristic of inorganic pigments:
 Weak colour strength
 Dull shades
 Low light fastness
 Low weather fastness

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 White pigment:
1. Titanium dioxide
2. Zinc oxide
3. Antimony oxide
4. White lead
5. Lead sulfate
 Colored pigments:
1. Iron oxide
2. Red lead
3. Cadmium red
4. Lead silicochromate
5. Lead chromates
6. Zinc chromates
7. Cadmium yellow
8. Calcium plumbate
• Metallic:
1. Aluminium
2. Zinc
3. Lead
• Extenders:
1. Blance fixe
2. Paris white
3. Barytes whiting

Natural pigments
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 Natural pigments are colored substances derived from natural sources, such as minerals, plants,
and insects.
 The colorants may be ground, washed, and sifted but otherwise are not chemically modified.
 Natural colors are pigments made by living organisms.
 Most common plant pigments are carotenoids, chlorophylls, anthocyanins and betalains
 Used as additives, color intensifiers, antioxidants, etc
 Natural colors will be used for the preservation of biodiversity.
 Example:
 Chlorophylls
 Carotenoids
 Flavonoides
 Anthocyanins
 Tannins
 Betalains
 Quinones

CHLOROPHYLL
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 It is the green coloured water insoluble pigment involved in photosynthesis are present in
many plants specially in green leafy vegetables. They also give the green colour to the "skin
of apples".
 Functional pigment of photosynthesis in green plants.
 Chlorophyll a: intense blue-green in colour
 Chlorophyll b: intense yellow green colour
Uses:
 The major portion of both oil-soluble and water- soluble forms is used in dairy products,
edible oils, soups, chewing gum, sugar confections, drinks

CAROTENOIDS
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 Carotenoid are also water insoluble pigment which is responsible for the -
Yellow, Orange, colour of fruits and vegetables widely distributed in nature.
 Carotenoids are extracted from Annatto, Saffron, Paprika, tomato etc.
 Some examples are:
 Yellow corn- cryptoxanthin
 Tomatoes- Lycopene
 Carrots- Beta carotene,
 Xanthophyll Red Capsicum- Beta Carotene
 Dietary source - Sweet potatoes, Carrots, Mangoes, Apricots, Spinach,, Beet,
Broccoli, Winter squash, Pumpkin.
 It is found in plants in two primary forms i.e a-carotene and B-carotene

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 E number of B-Carotene is E160a
 B-Carotene is the more common form and found in yellow, orange, and
green leafy fruits and vegetables
 Capable of producing colors from pale yellow to dark orange
Uses:
 Food coloring-juice, cakes, desserts, butter and margarine
 Maintenance of body health - antioxidant activities

ANTHOCYANINS
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 Anthocyanins are flavonoids commonly found in nature
 These are the red, blue, and violet water soluble compounds occuring in some fruits and
vegetables.
 Eg. Cherries, Red Apples, Blue Berries, Red grapes, Pomegranate are achieve their
colour & appeal because of anthocyanin.
 Pigments are extracted from red cabbage, strawberries, red rasp berries, blackberry,
cherry, etc…
Uses:
 Food colorings - Beverages, fruit fillings, Snacks, dairy products and confectionery

ANTHOXANTHINS
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 These are creamy yellow, creamy white or yellow white water soluble compounds occuring in the
cell sap of fruits & vegetables.
 They are colourless or white to yellow depending on pH.
 Pigments are generally whiter in an acid medium(lime juice or vinegar) or yellowed in an alkaline
medium.
Examples:
 Good sources of anthoxanthins include bananas, cauliflower, garlic, ginger, jicama, mushrooms,
onions, parsnips, potatoes, and turnips, Cabbage, etc…

BETALINS
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 Anthocyanin pigments are absent in some families which are replaced by
compounds Betacyanins and Betaxanthins together known as betalins.
 Betalin are water soluble pigment which are present in Beet root (beta vulgaris).
 Acidic medium promotes reddish colour whereas alkaline pH brings out brownish
blue.
 E number is E162.
Applications:
 Coloring agent in meat and sausages, ice cream and powdered soft drink
beverages, sugar confectionery,soups.
 Betanin absorbs well from the gut and acts as an antioxidant.

TURMERIC and SAFFRON
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 Turmeric is a root that has been used to impart colour and flavour to the
food, produced by rhizomes of perennial herb, Curcuma longa.
 The colouring responsible for the oleoresin "curcumin", it is stable to heat
but sensitive to alkali and light.
 Apart from colouring or flavour it has also medicinal property.
 Saffron is a spice derived from the flower of Crocus sativus, the crimson
stigma and styles, called threads are collected and dried to be used mainly
as seasoning & colouring agent(rich golden yellow colour due to crocin) in
food.

Example of Different types of pigments
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Pigment Colors Foods
Carotenoids Red – Yellow
Carrots, Squash, Tomatoes,
Peppers
Flavonoids White – Yellow White Cauliflower
Chlorophyll Green
Leafy Greens, Herbs, Green
Cabbage
Diarylheptanoids Yellow Turmeric

DYES
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 A dye is a natural or synthetic substance that is used to add colour in foods
and drinks.
 Dyes dissolve in water and are manufactured as powders, granules, liquids
or other special-purpose forms.
 They can be used in beverages, dry mixes, baked goods, confections, dairy
products, pet foods and a variety of other products.
 Types of dyes
1. Natural dyes
2. Synthetic dyes

Natural dyes
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 Colorants that impart colour it is added to food.
 They are available in many forms like liquids,powder,gels and pastes.
Exmples:
 Plants: indigo and saffron
 Insects: cochineal beetles and lac scale insects
 Animals: species of shellfish
 Minerals: ferrous sulphate,ochre and clay
 Others:
 caramel,annatto,beet powder,cochineal extract, beta-carotene,fruit
juice,vegetable juice,carrot oil,paprika,riboflavin,saffron,titanium
dioxide,tomato lycopene,turmeric.

Synthetic dyes
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 Also known as artificial colours.
 Manufactured by chemical reactions.
 Commonly used in food and pharmaceutical industries.
 Cheaper, stable and long shelf life compare to natural colour.
 These are the artificial, non- natural colours manufactured by chemical
reactions uses in industries (Foods,Pharmaceuticals)
 The synthetic dyestuff has many advantages over natural colours, that they
tended to be much brighter, more stable, cheaper and a wide range of shades are
available but have toxic properties also.
 Types:
1. Permitted synthetic dyes
2. Non permitted synthetic dyes

PERMITTED SYNTHETIC DYES
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 Permitted synthetic colours are safe to use in food and manufacturers use
them because they are less expensive and provide uniform and bright colours.
 They can also be blended to provide a number of secondary colours.
 The synthetic colors that are permitted to be used are mentioned below.
 Red from: Ponceau 4R, Azorubine and Erythrosine
 Yellow from: Tartrazine and Sunset Yellow FCF
 Blue from: Indigo Carmine and Brilliant Blue FCF
 Green from: Fast Green FCF

Ponceau 4R
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 Ponceau 4R is a synthetic colorant that may be used as a food
coloring.
 It is denoted by E Number E124. Its chemical name is 1-(4-
sulpho-1-napthylazo)- 2-napthol- 6,8-disulphonic acid,
trisodium salt.
 Ponceau is the generic name for a family of azo dyes.
 Ponceau 4R is a strawberry red azo dye which can be used in
a variety of food products and is usually synthesized from
aromatic hydrocarbons.
 It is stable to light, heat, and acid but fades in the presence of
ascorbic acid.
 It is used in Europe, Asia and Australia, but has not been
approved by the US FDA.

Sunset Yellow FCF
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 Sunset Yellow FCF is a petroleum-derived orange azo dye.
 The full chemical name of Sunset Yellow FCF is disodium 2-
hydroxy-1 (4sulfonatophenylazo) -naphthalene-6-sulfonate
 It has a pH dependent maximum absorption at about 480 nm
at pH 1 and 443 nm at pH 13.
 it is denoted by E Number E110.
 The acceptable daily intake (ADI) is 0-4 mg/kg.
 Sunset Yellow FCF has no carcinogenicity, genotoxicity, or
developmental toxicity in the amounts at which it is used.

Brilliant Blue FCF
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 Brilliant Blue FCF (Blue 1) is an organic compound classified as a
triarylmethane dye and a blue azo dye.
 It is denoted by E number E133.
 It is soluble in water
 the solution has a maximum absorption at about 628nm.

Fast Green FCF
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 Fast Green FCF is a sea green triarylmethane food dye.
 Its E number is E143.
 Fast Green FCF is poorly absorbed by the intestines.
 It is used as a quantitative stain for histones at alkaline pH after acid
extraction of DNA.
 Its absorption maximum is at 625 nm.

Permitted synthetic dyes used an
industries
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• E104: Quinoline yellow.
• E122: Carmoisine.
• E124: Ponceau 4R.
• E131: Patent blue V.
• E142: Green S.
• E122: Azorubine
• E102: Tartrazine
• E110: Sunset Yellow FCF
• E132: Indigo carmine
• E133: Brilliant Blue FCF
• E143: Fast Green FCF

Non permitted synthetic dyes
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 The non permitted synthetic dyes are also called as banned dyes.
 It is found that Amaranth, Rhodamine B are commonly used non-permitted
colours.
 It's used for food adulteration in sweets and confectionery.
 Rhodamine is known as a banned dye that is harmful to health because of its
causes carcinogen reproductive growth toxicity.
 The study showed that Rhodamine B, followed by Orange II and Metanil Yellow.
 were the two most common non-permitted or banned colours that were used in the
rhodamine B.
 It is important to note that Rhodamine B is a cancer causing colouring agent that
gives a pink colour.
 But is actually meant for use in the plastics and textile industry.

Examples of Non permitted synthetic
dyes
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 Metanil yellow
 Rhodamine B
 Amaranth
 Orange II
 Orange G
 Malachite green
 Acid magenta
 Butter yellow
 Blue VRS
 Auramine
 Sudan red colours

Methods of Analysis
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 Various analytical techniques are used to determine synthetic food colorants
in food samples, including spectrophotometry, thin layer chromatography,
capillary electrophoresis, high performance liquid chromatography and
mass spectrometry (MS).
 The two mainly used analytical methods are:
1. Spectrometric Methods (quantitative estimation)
2. Chromatographic Methods (qualitative estimation)

EXTRACTION AND ANALYSIS OF RHODAMINE AND
SUDAN IN CHILLY AND CURRY POWDER
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The presence of various non-permitted food colours in Chilli and curry samples were analysed using
 Preliminary colour test and
 UV spectrophotometric method
SPECIES ADULTRANT TEST INFERENCE
RHODAMINE Take 2g of sample in a test tube,
add 5ml of acetone
Appearance of red
colour
SUDAN 3 Take 1g of sample in a test tube
and add 2ml of hexane in it and
shake well. Let it settle and
decant the upper layer in another
test tube, add 2ml of acetonitrile
reagent and shake well.
Appearance of red
colour in the lower
acetonitrile layer.
Preliminary test

Weigh 2.5 grams of chilli powder sample
Transfer to 30 mL centrifuge tube
Add 20 mL of ethyl acetate and 10 mL of methanol
Blend with the homogenizer at 16,500 rpm for 5 minutes
Centrifuge for 5 minutes.
Filter the organic upper layer
Collect a 25 mL volumetric flask
Measure the absorbance
EXTRACTION PROCEDURE
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Preparation of standards of Sudan III dye:
 Standards of Sudan dye were prepared in the concentration range of 0.01-30
ppm from sudan stock solution.
Preparation of standards of rhodamine dye:
 Standards of each Rhodamine dye were prepared in concentration range of 1-
50mcg from Rhodamine stock solution.
UV SPECTROPHOTOMETRIC METHOD
 Spectrums were recorded. wavelength range: 400-700 nm.
 500 nm for Rhodamine and 520 nm for Sudan III were selected from
spectra due to their maximum absorbance at the respective wavelengths
 Absorbance of sample was measured

Spectrum absorption maxima of permitted
synthetic dyes
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s.no Name of dye Absorption maxima (nm)
1. Ponceau 4R 507
2. azorubine 516
3. erythrosine 527
4. tartrazine 427
5. Sunset yellow FCF 482
6. Indigo carmine 609
7. brilliant blue FCF 630
8. Fast green FCF 624

EXTRACTION AND ANALYSIS OF ANNATTO IN BUTTER AND ICE CREAM
BY HPLC METHOD.
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METHODS:
 Presence of annatto can be identified by
 Preliminary test
 HPLC method
CHEMICALS:
 Acetone, Acetonitrile (HPLC- grade), Trichloromethane, n-hexane, methanol,
hydrochloric acid, glacial acetic acid, ethanol 96%, petroleum ether, tert-butyl
methyl ether, diethyl ether, ammonia solution.

Preliminary test
37
SAMPLE RAPID TEST INFERRENCE
Ice cream It can be detected by placing ice
cream into a flat dish, adding
sodium bicarbonate solution and
then inserting a strip of filter paper.
After a few hours the paper is
stained brown in the presence of
annatto and turns pink on the
addition of a drop of stannous
chloride solution.
butter Butter+ 1-2 ml 10% sodium
hydroxide solution.
Appearance of yellow colour.

EXTRACTION OF BUTTER AND ICE CREAM
5 g of the sample and
ethanol-water-ammonia solution+ petroleum ether (15 ml)
shake for 1 min. centrifuge for 5 min
remove and discard the ether layer
The extraction was repeated twice.
Add 11% aqueous acetic acid (10 mL) and n-butyl methyl ether (10 mL) and then shake
for 1 minute
Transfer the upper layer into a 100 mL conical flask
The extraction was repeated twice.
which can be broken by addition of ethanol
The collected extracts were evaporated at 40 °C.
Residue was dissolved in methanol/glacial acetic acid (10 mL,7:3 (v/v)).
EXTRACTION OF BUTTER AND ICE
CREAM
38

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 HPLC method:
✓solvent mix --Methanol : acetic acid (5%) 75:25 (v/v)
✓Isocratic flow-- 1 mL/min
✓Column temperature-- 40 °C
✓Detection wavelength --460 nm
✔Injection volume --10 µL.
 RESULT:
Elution time of the components were noted and the chromatogram was
compared with standard.

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THANK YOU

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