PIGMENTS AND DYES, food analysis, analysis

m.pharm analysis, pharmaceutical analysis, food analysis, cosmetic analysis and evaluation, modern bio analytical techniques, advanced pharmaceutical analysis, phamaceutical validation

1. 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 1 ST SEMESTER DEPT.OF PHARMACEUTICAL ANALYSIS KARNATAKA COLLEGE OF PHARMACY BANGALORE SUBJECT: FOOD ANALYSIS

2. INTRODUCTION OF PIGMENTS KARNATAKA COLLEGE OF PHARMACY 2 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.

3. KARNATAKA COLLEGE OF PHARMACY 3 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 . 

4. Characteristic of pigment KARNATAKA COLLEGE OF PHARMACY 4 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

5. Properties of Pigments KARNATAKA COLLEGE OF PHARMACY 5 No auxochrome group. Applicable to all kinds of fibers or textile materials. Rubbing fastness is poor. Particle size: 0.2-0.4 μ m 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.

6. KARNATAKA COLLEGE OF PHARMACY 6 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 Organic pigments Inorganic pigments

7. KARNATAKA COLLEGE OF PHARMACY 7 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.

8. KARNATAKA COLLEGE OF PHARMACY 8 Characteristic of organic pigments: High colour strength Give bright shades High light fastness Good weather fastness Types of organic pigments: Azo pigment Non azo pigments Polycyclic Dioxazine Anthraquinone Quinophthalone Triaryl Carbonium

9. KARNATAKA COLLEGE OF PHARMACY 9 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

10. KARNATAKA COLLEGE OF PHARMACY 10 White pigment: Titanium dioxide Zinc oxide Antimony oxide White lead Lead sulfate Colored pigments: Iron oxide Red lead Cadmium red Lead silicochromate Lead chromates Zinc chromates Cadmium yellow Calcium plumbate Metallic: Aluminium Zinc Lead Extenders: Blance fixe Paris white Barytes whiting

11. Natural pigments KARNATAKA COLLEGE OF PHARMACY 11 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

12. CHLOROPHYLL KARNATAKA COLLEGE OF PHARMACY 12 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

13. CAROTENOIDS KARNATAKA COLLEGE OF PHARMACY 13 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

14. KARNATAKA COLLEGE OF PHARMACY 14 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

15. ANTHOCYANINS KARNATAKA COLLEGE OF PHARMACY 15 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

16. ANTHOXANTHINS KARNATAKA COLLEGE OF PHARMACY 16 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 …

17. BETALINS KARNATAKA COLLEGE OF PHARMACY 17 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.

18. TURMERIC and SAFFRON KARNATAKA COLLEGE OF PHARMACY 18 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.

19. Example of Different types of pigments KARNATAKA COLLEGE OF PHARMACY 19 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

20. DYES KARNATAKA COLLEGE OF PHARMACY 20 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 Natural dyes Synthetic dyes

21. Natural dyes KARNATAKA COLLEGE OF PHARMACY 21 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 .

22. Synthetic dyes KARNATAKA COLLEGE OF PHARMACY 22 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: Permitted synthetic dyes Non permitted synthetic dyes

23. PERMITTED SYNTHETIC DYES KARNATAKA COLLEGE OF PHARMACY 23 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

24. Ponceau 4R KARNATAKA COLLEGE OF PHARMACY 24 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.

25. Sunset Yellow FCF KARNATAKA COLLEGE OF PHARMACY 25 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.

26. Brilliant Blue FCF KARNATAKA COLLEGE OF PHARMACY 26 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.

27. Fast Green FCF KARNATAKA COLLEGE OF PHARMACY 27 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.

28. Permitted synthetic dyes used an industries KARNATAKA COLLEGE OF PHARMACY 28 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

29. Non permitted synthetic dyes KARNATAKA COLLEGE OF PHARMACY 29 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 . B ut is actually meant for use in the plastics and textile industry.

30. Examples of Non permitted synthetic dyes KARNATAKA COLLEGE OF PHARMACY 30 Metanil yellow Rhodamine B Amaranth Orange II Orange G Malachite green Acid magenta Butter yellow Blue VRS Auramine Sudan red colours

31. Methods of Analysis KARNATAKA COLLEGE OF PHARMACY 31 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: Spectrometric Methods (quantitative estimation) Chromatographic Methods (qualitative estimation)

32. EXTRACTION AND ANALYSIS OF RHODAMINE AND SUDAN IN CHILLY AND CURRY POWDER KARNATAKA COLLEGE OF PHARMACY 32 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

33. 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 KARNATAKA COLLEGE OF PHARMACY 33

34. KARNATAKA COLLEGE OF PHARMACY 34 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

35. Spectrum absorption maxima of permitted synthetic dyes KARNATAKA COLLEGE OF PHARMACY 35 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

36. EXTRACTION AND ANALYSIS OF ANNATTO IN BUTTER AND ICE CREAM BY HPLC METHOD . KARNATAKA COLLEGE OF PHARMACY 36 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.

37. Preliminary test KARNATAKA COLLEGE OF PHARMACY 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 .

38. 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 KARNATAKA COLLEGE OF PHARMACY 38

39. KARNATAKA COLLEGE OF PHARMACY 39 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.

40. KARNATAKA COLLEGE OF PHARMACY 40 THANK YOU