QUALITY ASSESSMENT OF ALLURA RED IN LOCAL CONFECTIONERIES BY USING TLC 198120022987000 Submitted By Miss ANAM 2012- MS-APP- CHEM- 27 Thesis Supervisor Dr

QUALITY ASSESSMENT OF ALLURA RED IN LOCAL CONFECTIONERIES BY USING TLC
198120022987000

Submitted By
Miss ANAM
2012- MS-APP- CHEM- 27
Thesis Supervisor
Dr. Arjumand I. Durrani(Associate Professor)

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University of Engineering and Technology
Lahore, Pakistan – 2014
QUALITY ASSESSMENT OF ALLURA RED IN LOCAL CONFECTIONERIES BY USING TLC
A RESEARCH THESIS SUBMITTED
TO
THE UNIVERSITY OF ENGINEERING AND TECHNOLOGY
LAHORE In partial Fulfillment of the Requirements
For The Degree of Master of Science
In
Applied Chemistry
2012-2014
By
MISS ANAM
2012- MS-APP- CHEM- 27
DEPARTMENT OF CHEMISTRY
University of Engineering and Technology
Lahore, Pakistan-2014
QUALITY ASSESSMENT OF ALLURA RED IN LOCAL CONFECTIONERIES BY USING TLC
This research Thesis is submitted to the Department of Chemistry, University of Engineering and Technology Lahore – Pakistan, for the partial fulfillment of the requirements for the degree of
Master in Science
In
Applied chemistry
4304665196840006222900
Project supervisor External Examiner
Dr. Arjumand I.Durrani175958514287400
Prof. Dr. Syeda Rubina Gilani(Chairperson, Dept. of Chemistry)
DEPARTMENT OF CHEMISTRY
University of Engineering and Technology
Lahore – 2014
DEPARTMENT OF CHEMISTRY
University of Engineering and Technology
Lahore
Form for Release of Thesis for Examination
I, ANAM declare that the Thesis titled:
“QUALITY ASSESSMENT OF ALLURA RED IN LOCAL CONFECTIONERIES BY USING TLC” is submitted for the partial fulfillment of the requirements for the degree of Master of Science in Applied Chemistry.

Signature of Candidate_________________
Date _______________
It is approved that the above Thesis be submitted for examination.

Signature of Supervisor
Internal Examiner __________________date _______________
This form must be submitted with the Thesis to the chairperson, Department of Chemistry University of Engineering & Technology, Lahore-Pakistan.

DEDICATED
TO MY EVER BELOVING AND KIND PARENTS, SISTERS AND BROTHER WITHOUT THEIR SUPPORT, I AM UNABLE TO DO ANY JOB
ALSO DEDICATED TO MY SUPERVISOR
Dr. ARJUMAND I. DURRANI
WHO HELPED ME IN EVERY WAY
ACKNOWLEDGEMENT
In The Name Of ALLAH The Most Beneficial, Merciful, Compassionate The Almighty Creator Of The Whole Universe. Peace be upon His Messenger THE HOLY PROPHET MUHAMMAD, The Eternal Source of guidance for all the times.

It is great privilege that I have worked under the kind meritorious supervision of Dr. ARJUMAND I. DURRANI for my research Thesis on “QUALITY ASSESSMENT OF ALLURA RED IN LOCAL CONFECTIONERIES BY USING TLC” for the award of degree of Master of Science in the subject of Applied Chemistry. I am highly obliged for her scholarly, ever encouraging guides, valuable pieces of advice and suggestions.It is my pleasant duty to record my sincerest gratitude to Prof. Dr. Syeda Rubina Gilani for providing me the opportunity to do this work. May Allah bless her in every part of their life (Ameen).

I am also highly thankful to my friends for guidance in this regard and help me in every problem I faced during my research work. I acknowledge the respective cooperation of the whole laboratory staff and express my thanks to all of them.

ANAM
2012-MS-APP-CHEM-27

ABSTRACT
ALLURA RED is a synthetic food dye used in food products. The aim of this work is the analysis of allura red in food products. 16 samples of branded and non-branded food products were collected. The extraction of color was done by using wool extraction method. Separations and identification of allura red carried out by Thin Layer Chromatography by using silica gel GF254 as adsorbent and Propanol: Ammonia (4:1) as a solvent, then followed by analysis using UV spectrophotometer. The standard reference values range for allura red are Rf =0.51-0.60 and lambda max =504nm.TLC and UV spectrophotometric results showed that nearly all the samples collected from high to medium standard shops had permitted colors which were within the prescribed limits however samples which were unwrapped and were collected from low standard shops contained non-permitted colors which are harmful and carcinogenic for human health.
TABLE OF CONTENT
Chapter No. Topics Pg.#
1 Introduction 11
1.1 Food Colors 12
1.2 Allura Red 20
2 Aim of The Work 25
3 Literature Survey 27
4 Theoretical Background 33
4.1 Thin Layer Chromatography 34
4.2 Spectrophotometer 37
5 Experimentation 41
5.1 Sample Collection 43
5.2 Sample Preparation 45
5.3 Sample Analysis 47
6 Results & Discussion 55
7 Summary & Conclusion 56
8 References 58
List of Figures
Fig. No. Description Page No.

1.1 The Structural Formula of Allura Red AC 22
4.1 TLC 33
4.2 Steps to prepare TLC Plates 34
4.3 Development of TLC plates preparation 35
4.4 Spectrophotometer 36
4.5 Principle of Spectrophotometer 37
6.1 Allura Red Dye 48
6.2 Custard Strawberry 48
6.3 Delve Mango Custard 48
6.4 Delve Orange 48
6.5 Delve Vanilla 48
6.6 Gardens Falsa 48
6.7 Jaam e sheren49
6.8 National Jelly Crystals 49
6.9 Sharbat e Jann49
6.10 Sting 49
6.11 Tang Freeze 49
6.12 Tang Mango 49
6.13 Tang Strawberry 50
6.14 Vimto 50
List of Tables
Table No. Description Pg.#
1.1 Description of Natural Food Dyes 14
1.2 Name,Shade,E-no, and FD & C No of Synthetic Food Colors 15
1.3 Properties of allura red dye 22
5.1 Detail of analyzed confectioneries samples 42
6.1 Comparison b/w food colors stated on the label & identified by TLC 47
6.2 Rf value and lambda max of various confectioneries samples 51
CHAPTER NO.1
INTRODUCTION

Chapter01 Introduction
CHAPTER 01 INTRODUCTION
FOOD COLOR
Contains the chemical used to include color to eat the color of the food. They are used to maintain or improve food appearance. These compounds are used in many forms, including alcohol, powder, gel, and pastor etc. Both food and tea are used in food and home cooking. 1 Due to its safety and general availability, food is also used in non-food applications, including cosmetics, pharmaceuticals, home craft projects and medical equipment.

The FDA has assured color extra organizations that they are safe for human use. Regulation also ensures that food can be labeled properly with color so that consumers know what they eat. 2 To determine an additional approval, the FDA study its structure and how much of it is consumed and remembers any health effects and safety factors that need to be observed. After approving food color, the FDA uses the appropriate surface for this extra. Allows the FDA to approve only an extra if the customer is sure to have any harm. 3
Types Of Color Additives
Certification of Color Additives
All color extra items listed by the FDA need to fall into two types: those under the FDA certification process and those who are exempted from the certification process. Extra colored nutrients of batch certification are artificial organic colors, lakes or sunshine. Those for food use are chemically classified as azo, xanthene, triphenylmethane, and indigoid dyes. Although static color extra items are called coal tar colors due to their traditional population, they are now synthesized with raw materials obtained from petroleum 4.

Exempt From Certification of Color Additives
Chapter01 Introduction
Color additives exempt from certification generally include those derived from plant or mineral sources. One, cochineal extract (and its lake, carmine) is derived from an insect. Most are straight colors; one exception is carmine as described above. Certification exempt color additives must comply with the identity and purity specifications and use limitations described in their listing regulations. Users of these color additives are responsible for ensuring that the color additives comply with the listing regulation. There are two types of approved color additives 5.

Dyes
Lakes
Colors are water-dissolvable and normally come as powders, granules, or fluids. Lakes are not water-solvent. They are found in items containing fats and oils. Some nourishment colorings are artificially delivered. Cases of these shading added substances incorporate FD&C Blue No. 1 and 2 and FD&C Green No. 3. Other nourishment colorings originate from shades of vegetables, minerals, or animals. 4 Examples of these common added substances incorporate beta-carotene, grape skin extricate, caramel shading, and saffron. Colors break down in water, yet are not solvent in oil. Colors are fabricated as powders, granules, fluids or other unique reason shapes. They can be utilized in refreshments, dry blends, prepared merchandise, sugary treats, dairy items, pet nourishments, and an assortment of different items. Colors additionally have symptoms which lakes don’t, including the way that a lot of colors ingested can shading stools. 6
Lakes are made by consolidating colors with salts to make insoluble mixes. Lakes tint by scattering. Lakes are not oil dissolvable, but rather are oil dispersible. Lakes are steadier than colors and are perfect for shading items containing fats and oils or things lacking adequate dampness to break up colors. Normal uses incorporate covered tablets, cake and donut blends, hard confections and biting gums, lipsticks, cleansers, shampoos, powder, etc. 7
Chapter01 Introduction
Natural Food Dyes
A growing number of natural food dyes are being commercially produced, partly due to consumer concerns surrounding synthetic dyes. Some example include
Table 1.1. Description of Natural Food Dyes
Colorant Color Uses
Anthocyanin Blue-reddish shades Soft drinks ,alcoholic drinks ,pickles
Annatto Orange shade Dairy ; fat products ,desserts
Beta-carotene Yellow orange Butter ,fats ,oils ,soft drink, fruit juices ,ice creams
CanthoxanthinOrange-red-red souses, soups ,meat ;fish dishes
Paprika Orange red Meat products ,snack ,soups ,salad
Saffron Yellow Baked goods ,rice dishes ,meat dishes ,soups
CrocinYellow Dairy products ,jams ,pasta ,rice
LucinYellow ice creams ,dairy products ,sugar ,flour
Beet powder Bluish red Frozen, ice creams ,flavored milk
Cochineal Orange soft ,alcoholic drinks
Carmine Bluish red Soft drinks ,sugar ;flavor confectionary ,pickles, souses
Sandal wood Orange-orange red Fish processing ,alcoholic drinks ,sea food dressings, meat products
Chlorophyll Olive Green Soups ,souses ,fruit products ,jams
Chapter01 Introduction
To guarantee reproducibility, the shaded segments of these substances are regularly given in exceedingly decontaminated shape, and for expanded solidness and comfort, they can be planned in appropriate transporter materials (strong and fluids). Hexane, CH3)2CO and different solvents separate cell dividers in the products of the soil and take into consideration most extreme extraction of the shading. Hints of these may at present stay in the completed colorant, yet they don’t should be proclaimed on the item mark; this is on account of they are a piece of a gathering of substances known as persist fixings. Nourishment colorants can some of the time cause hypersensitive responses and anaphylactic stun in touchy people. Characteristic shading specialists known to be potential dangers incorporate annatto, cochineal and carmine. 8
Artificial Food Color Artificial Food
s
1.1.4. Purpose of Food Coloring
Individuals connect certain hues with specific flavors, and the shade of nourishment can impact the apparent flavor in anything from treat to wine.
Chapter01 Introduction
A few times the point is to invigorate a shading that is seen by the shopper as regular, for example, adding red shading to glace fruits yet here and there it is for impact, similar to the green ketchup that Heinz propelled in 19999. Color additives are used in foods for many reasons including:
•Offset color loss due to exposure to light, air, temperature extremes, moisture and storage condition.

•Correct natural variations in color
•Enhance colors that occur naturally
•Provide color to colorless and “fun” foods
•Make food more attractive and appetizing, and informative
•Allow consumers to identify products on sight, like candy flavors or medicine dosages.10
Since sustenance colors are for the most part more secure to use than ordinary specialists’ colors and shades, a few craftsmen have utilized nourishment shading as a methods for making pictures, particularly in structures, for example, body-painting. Red nourishment color is frequently utilized in dramatic blood. Most counterfeit sustenance colorings are a kind of corrosive color, and can be utilized to color protein strands and nylon with the expansion of a corrosive. They are all wash quick and most are additionally lightfast. They won’t forever cling to plant filaments and other synthetic. 11
History Of Food Dyes
In naturally coming colors, automotive used in vegetables and minerals used to color food, drugs, and cosmetics in ancient times. Paprika, turmeric, saffron, iron and lead oxide, and copper sulfate are some examples. Artificial colors used in early Egyptian cosmetics and hairstyles. Alcohol was artificially colored at least 300 BC by alcohol. 12
In 1856, William Henry Perkin discovered the first synthetic organic dye, called mauve. Discoveries of similar dyes soon followed and they quickly became used to color foods, drugs, and cosmetics. Because these dyes were first produced from by-products of coal processing, they were known as “coal-tar colors. “Federal oversight of color additives began in the1880s. The
Chapter01 Introduction
assessment of color-imparting ingredients in foods was among the first public initiatives undertaken by the U.S. when, in 1881, the U.S. Department of Agriculture’s (USDA) Bureau of Chemistry began research on the use of colors in food. Butter and cheese were the first foods for which the federal government authorized the use of artificial coloring. 13
Food and Drug Act. In 1906, the Congress Food and Drug Act passed, which prohibited use of toxic or extruded colors in the confectionery, and prevented eating or painting of harm or damage to hide the waist. USDA was the initial enforcement option for this process. In 1907, USDA, which included a list of seven direct colors, was approved for use in food. Later, the FID established voluntary certification programs in the early part of the century and entered into new colors. In 1927, the responsibility of enforcement of Food and Drug Act was given to the newly created FDA of 1906. 14 (The agency first invited the food, drugs and insect counter-terrorism administration and its current name was given in 1930.) By 1931, 15 direct colors were approved for use in food. Given that six out of seven uses include: FDD and Blue N1 (Brilliant Blue FFF), FD ; C Blue No 2 (Ingogenone), FD and C Green Number 3 (Fast Green F CF), FD and C Red No 3 (Orthroscience), FD and C Yellow No 5 (Tetrrazen), and FD and C Yellow No 6 (Sunset Pale) .Doodland Food, Drugs, and Cosmetic Act 19 38 In 1938 and 1930, 15 it became clear that the 1906 Food and Drug Act did not run adequately to protect public health, adultery, and even poisonous products, including a mahram which Some ladies kept blind. 1938 of Federal Food, Drugs, and Cosmetic Act increased the monitoring of food and drugs, and for the first time, the law passed the rules for cosmetics and medical instruments. 16

Chapter01 Introduction
400050-628650 History of Food Dyes
History of Food Dyes
962025-12382500 Coloring of candies (Egyptians,1500.B.C)
Wine is colored (400.B.C)
10477508191500Early civilization
Natural colorants are used to pigment man-made products.
Saffron ;other plant species are employed in the pigmentation
Of butter.

Use of inorganic pigments (e.g. lead chromate, copper sulphate) in the
Production of candies and sauerkraut.

W.H.Perkin synthesis mauve or aniline purple form coal tar distillates(1856)
10096508763000 19th century Diazonium coupling reaction by Peter Giess (1860)
Para red, lethal red and hansa yellow pigments are synthesized (1895)
Use of tar colorants and other petroleum derivatives in food, medicine
and cosmetics.
10096508953500 1905 Red 3 (toluidine red is synthesized)
Around 200 synthetic colorants are provisionally listed by FDA and remained
10001258572500 1938
Up to 1960.

Attack to food additives. The main target fast food but colorants
9906009906000 1960-1970 are the best Sword.

Natural colorants market is valued at $250 million(US).

9810758382000 1994
The annual growth of the natural colorants market is valued over
9810757048500 2000
$1 billion (US)
Chapter01 Introduction
Regulation of Color Additives
FDA has administrative oversight for shading added substances utilized in nourishments, medications, beautifying agents, and therapeutic gadgets. FDA records new shading added substances or new uses for recorded shading added substances that have been appeared to be alright for their planned uses in the Code of Federal Regulations (CFR), conducts an affirmation program for clumps of shading added substances that are required to be ensured before deal, and screens the utilization of shading added substances in items in the U.S., including item naming. These exercises originate from FDA’s part in authorizing the shading added substance arrangements of the FD&C Act, the Fair Packaging and Labeling Act, and other pertinent laws, including the as of late established Public Health Security and Bioterrorism Preparedness and Response Act of 2002 that requires local and remote makers of shading added substances utilized as fixings in nourishments to enroll with FDA by December 12, 2003. 17
Uses Of Food Color
There are vast used of food colors and lakes. Today these colors are used in many applications, some of which are listed below:
Soft drink & Beverages, Alcoholic & Non – Alcoholic Drinks, Desserts, Edible Ices & Confectionery, Decorations & coatings, Jams & jellies, Desserts & Dry mixes ,Baked Goods, Pickles, Sauces & Seasonings, Canned Products, Toothpaste, Meat & Fish products, Sausages, Cheese, Tablets & Capsules, Syrups, Cosmetics, Bath Salts, Hair rinses, Shampoos, Pet Foods, Animal Feeds, Toiletry products, Washing powders, Soft Toys, Writing Inks, Pesticides, Coated candies, Chewing Gum, Gum Salts, Summer Coatings, Wafers, Yoghurts, Ice Creams, Lotions, Lipsticks, Eyeliners,& Eye shadows, Plastic film, Snacks powder, Dry Beverages &
Dessert powder, & spice compounds.18
Side Effect Of Food Dyes
Since it is an azo color, it might inspire narrow mindedness in individuals bigoted to salicylates. Also, it is a histamine hero, and may increase side effects of asthma. In blend with benzoates, it is likewise embroiled in hyperactivity in kids.
Chapter01 Introduction
One of its degradation products causes bladder disease in creatures when show in high fixations. In the late 1800s, a few makers shaded items with conceivably noxious mineral and metal based mixes. Lethal synthetic substances tinted certain confections and pickles, while other shading added substances contained arsenic or comparable toxins. Authentic records demonstrate that wounds, even passing’s, came about because of polluted colorants .Food makers additionally tricked clients by utilizing shading added substances to cover poor item quality or ruined stock.1819
By the turn of the century, unmonitored shading added substances had spread through the commercial center in a wide range of prominent sustenance’s, including ketchup, mustard, jams, and wine. Vender at the time offered in excess of 80 fake shading specialists, some proposed for coloring materials, not nourishments. Many shading added substances had never been tried for danger or other unfriendly impacts. 20
ALLURA RED AC
1.2.1. General Properties of Allura Red AC
Allura Red AC is a red azo dye that goes by several names including: Allura Red, Food Red 17, C.I. 16035, FD&C Red 40,E129,910 2-naphthalenesulfonic acid, 6-hydroxy-5-((2-methoxy-5-methyl-4-sulfophenyl )azo)-, disodium salt, and disodium 6-hydroxy-5-((2-methoxy-5-methyl-4-sulfophenyl)azo)-2-naphthalenesulfonate. It is used as a food dye and has the E number E129. Allura Red AC was originally introduced in the United States as a replacement for the use of amaranth as a food coloring21 It has the appearance of a dark red powder. It usually comes as a sodium salt, but can also be used as both calcium and potassium salts. It is soluble in water and slightly soluble in 50% ethanol, its maximum absorbance lies at about 504 nm. Its melting point is above 300 degrees Celsius. Allura Red AC is one of many High Production Volume Chemicals. Red AC was originally manufactured from coal tar, but is now mostly made from petroleum. Despite the popular misconception, Allura Red AC is not derived from any insect, unlike the food coloring carmine, which is derived from the female cochineal insect.2122
Chapter01 Introduction
Table 1.3 General Properties of Allura Red Dye
Molecular formula C18H14N2Na2O8S2
Molar mass 496.42gmol-1
Appearance Red powder
Melting point >300 C
Registry Number 25956-17-6
IUPAC name Disodium 6- hydroxyl-5-((2-methyl-4-
Sulfophenyl)azo)-2-naphthalenesulfonate
Other names Allura Red,Food Red 17,C.l. 16035,FD&C Red 40,E129,2-naphthalenesulfonic acid disodium salt
Merck Index Number 284
Dye Class Azo
Solubility Soluble in water, ethanol
Certification Approved by Food & Drugs Administration
1.2.2. Structure of Allura Red AC

Figure: 1.1 The structural formula of Allura Red AC
Chapter01 Introduction
At least 17 synonyms are in use ( ChemIDplus advanced, via internet,2006).The most commonly used synonyms in published literature are Allura Red AC, Food Red No. 40 and FD&C Red No.40. In the past, the synonym Red Z-4576 was frequently used in unpublished literature.23
1.2.3. Potential Behavioral Effects
A 2007 report from Southampton University questioned the safety of azo food dyes in three year old and 8-9 year old children. In a 2006 report, the authors had noted it was difficult to eliminate all these dyes without causing nutritional deficiencies. 24Professor Stevenson, a co-author of both reports, said, “This has been a major study investigating an important area of research.” The results suggest that consumption of certain mixtures of artificial food colors and sodium benzoate preservative are associated with increases in hyperactive behavior in children. “However, parents should not think that simply taking these additives out of food will prevent hyperactive disorders. We know that many other influences are at work but this at least is one a child can avoid.25
1.2.4. Regulation of Allura Red AC
In Europe, Allura Red AC is not recommended for consumption by children. It is banned in Denmark, Belgium, Fre and Switzerland, and was also banned in Sweden until the country joined the European Union in 1994. The European Union approves Allura Red AC as a food colorant, but EU countries’ local laws banning food colorants are preserved. In Norway, it was banned between 1978 and 2001, a period in which azo dyes were only legally used in alcoholic beverages and some fish products. 2526
Application Of Allura Red AC
In the United States, Allura Red AC is approved by the Food and Drug Administration (FDA) for use in cosmetics, drugs, and food. It is used in some tattoo inks and is used in many products, such as soft drinks, children’s medications, and cotton candy. It is by far the most commonly used red dye in the United States, completely replacing amaranth (Red 2) and also replacing erythrosine (Red 3) in most applications due to the potential health effects of the two dyes.32
Chapter01 Introduction
On June 30, 2010, the Center for Science in the Public Interest (CSPI) called for the FDA to ban Red 40.33
Staining Application
Alcohol, beverage, drink, syrup, candies, cotton candies, baked food, frozen food, aerated fruits, sea food,sweetner,capsules,pharmaceutical dosage, tablets, toothpaste,sunscreen,dental bleaching gel, eye brows,lips,skin,body surfaces,tattoos,hair,Keratin fibres,cocoa bath, waters.26
Biological Application
Antifungal formulation, treating bone metabolic diseases, coughing, sneezing, rhinorrhea, nasal obstruction, periodontal disease, rhinitis. 26
Industrial Application
Antireflective coatings, inks, detergents, colored bubbles, fibric softners, textiles, entertainment product, toys 26
Limitation
Carcinogenicity
Chromosomal aberration
Developmental toxicity
DNA damage
Genotoxicity
Hyperactive behavior in children
Mutagenicity
Neurotoxicity
Psych toxicity
Reproductive toxicity27

CHAPTER No. 2
AIM OF THE WORK
Chapter 02 Aim Of The Work
2.1. AIM OF THE WORK
Any nutritious substance that individuals or creatures eat or drink or that plants assimilate keeping in mind the end goal to keep up life and development is said to be sustenance thusly we are essentially worry with nourishment science, Food science manages the investigation of compound procedures and associations of all organic and non-natural segments of sustenance’s. The organic substances incorporate such things as meat, poultry, lettuce, lager, and drain as illustrations. In Pakistan, such as creating nations, nourishment items are prepared both in mechanical and non-modern area. There is quality check in mechanical division yet there is no quality control in non-modern segment.
Food color makers play an important role in eating food or extra items, on the other side they have some limit. In order to ensure the health effects, the color of this diet should be in the range of permissible. Therefore, many manufacturers and street buyers produce non-standard food products. Food color is one of the main causes of poisonous foods. It is an extraordinary trend that people are attracted specifically to colorful foods and drinks because it is attractive and hungry. Artificial food is used in food products. But if they use too much, they are not safe for human health.
Many surveys have been made to identify food colors present in food products. The technique used for this purpose is the carousel zone electrophorescence, HPLC, RPHPLC, HPLC diode art detector, paper chromatography. This technique is used for separation techniques. HPLC is a technique used to separate ingredients in a mixture, to identify every component and in each component in quantity.

In this research work, TLC has been used for the identification of Allura Red Dye in jellies, custard, Tang, soft drink followed by UV spectrophotometer.

CHAPTER NO. 3
LITERATURE SURVEY
Chapter 03 Literature Survey
S.S.chou, YH Lin et al. 28 chipped away at technique for micellar electrokinetic slim chromatography (MEKC) was produced for synchronous assurance of 14 manufactured hues in soda pops and sweet shops. The ideal dissolvable of MEKC technique for isolating all hues was a blended arrangement contained 18% acetonitrile and 82% 0.05 M sodium deoxycholate in borate-phosphate cradle (pH 7.8). These hues were very much isolated inside 20 min utilizing 57 cm × 75 micrometer uncoated combined silica slender section, working at 25 kV and recognized by UV at 214 nm. The normal recuperation of all hues spiked into soda pops and candy parlor was superior to 82%. The expansion of unlawful hues was not found subsequent to testing 30 tests. In retail nourishments, the colorant found in most elevated focus was tartrazine (155 ?L/g test)
S.chanlon, L. Joly 29 took a shot at Carmoisine (E 122), Ponceau 4R (E 124) and Allura red (E 129), are manufactured azo colors 7generally used to give red shading to syrups, pop and desserts. They could be effortlessly recognized from the regular colors, which are not electroactive, utilizing differential heartbeat polarography. The impact of the pH on the forces and the possibilities of the pinnacles was examined between pH 3 and 11, and acidic or firmly fundamental media showed up not helpful. It was demonstrated that in a pH=9 phosphate cushion, the pinnacles of Carmoisine, Allura red and Ponceau 4R were very much molded and isolated, permitting precise recognizable proof and measurement, regardless of whether the three colors were blended. No critical changes of the pinnacle possibilities were seen in the business tests, and subsequently the colors can be recognized without vagueness. A method utilizing the standard expansion strategy was approved with test syrups. The recuperation was in the 96– 105% territory and the relative standard deviation was near 1% for the three colors. The breaking points of evaluation in the polarographic cell, assessed from the polarographic information, were 42, 43 and 34 ?g L?1 for Carmoisine, Allura red and Ponceau 4R, separately. The strategy was connected to business sodas and desserts. The outcomes were compared to those obtained using liquid chromatography, and they appeared to be in good agreement.

Katerina S. Minioti 30 worked on reversed-phase high performance liquid chromatographic method for the successful separation and determination of 13 synthetic food colorants (Tartrazine E 102, Quinoline Yellow E 104, Sunset Yellow E 110, Carmoisine E 122, Amaranth E 123, Ponceau 4R E 124, Erythrosine E 127, Red 2G E 128, Allura Red AC E 129, Patent Blue V E
Chapter 03 Literature Survey
131, Indigo Carmine E 132, Brilliant Blue FCF E 133 and Green S E 142) was developed. A C18 stationary phase was used and the mobile phase contained an acetonitrile–methanol (20:80 v/v) mixture and a 1% (m/v) ammonium acetate buffer solution at pH 7.5. Successful separation was obtained for all the compounds using an optimized gradient elution within 29 min. The diode-array detector was used to monitor the colorants between 350 and 800 nm. The method was thoroughly validated. Detection limits for all substances varied between 1.59 (E 142) and 22.1 (E 124) ?g L?1. The intra-day precision (as R.S.D.r) ranged from 0.37% (E 122 in fruit flavored drink at a concentration of 100 mg L?1) to 4.8% (E 142 in icing sugar at a level of 0.9 mg kg?1). The inter-day precision (as R.S.D.R) was between 0.86% for E 122 in fruit flavored drink at 100 mg L?1 and 10% for E142 in jam at a concentration of 9 mg kg?1. Satisfactory recoveries, ranging from 94% (E 142 in jam) to 102% (E 131 in sweets), were obtained. The method was applied to the determination of colorants in various water-soluble foods, such as fruit flavored drinks, alcoholic drinks, jams, sugar confectionery and sweets, with simple pre-treatment (dilution or water extraction).

Hao Wv, Jing –bo Gvo et al. 31 worked on novel and simple rapid shaking-based method of ionic liquid dispersive liquid phase microextraction for the determination of six synthetic food colorants (Tartrazine, Amaranth, Sunset Yellow, Allura Red, Ponceau 4R, and Erythrosine) in soft drinks, sugar- and gelatin-based confectionery was established. High-performance liquid chromatography coupled with an ultraviolet detector was used for the determinations. The extraction procedure did not require a dispersive solvent, heat, ultra sonication, or additional chemical reagents. 1-Octyl-3-methylimidazolium tetrafluoroborate (C8MIMBF4) was dispersed in an aqueous sample solution as fine droplets by manual shaking, enabling the easier migration of analyses into the ionic liquid phase. Factors such as the C8MIMBF4 volume, sample pH, extraction time, and centrifugation time were investigated. Under the optimum experimental conditions, the proposed method showed excellent detection sensitivity with limits of detection (signal-to-noise ratio = 3) within 0.015–0.32 ng/mL. The method was also successfully used in analyzing real food samples. Good spiked recoveries from 95.8%–104.5% were obtained.

M.G.Kiseleva et al.32 worked on retention of synthetic food dyes on the sorbent Kromasil C18 was studied under the conditions of reversed-phase and ion-pair high-performance liquid
Chapter 03 Literature Survey
chromatography, depending on the concentrations of the ion-pair reagent (tetrabutylammonium dihydrophosphate) and organic solvent (acetonitrile) in the mobile phase and on the concentration and pH of the phosphate buffer solution. The optimum conditions were selected for the separation of ten dyes in the isocratic elution mode within 25 min. A procedure was developed for the determination of synthetic food dyes in foodstuffs; the detection limit was 10–30 ?g/L.

Ates,Ebrui et al.33 worked on cloud point extraction method is reported using LC/MS for the determination of regulated water-soluble food colors (Allura Red, Sunset Yellow, erythrosine, and tartrazine) and banned fat-soluble synthetic azo dyes (Sudan I, II, III, and IV; Red B; 7B; Black B; Red G; Metanil Yellow; and Rhoda mine B). The extraction of all 14 colors was carried out with cloud point extraction using the nonionic surfactant Triton X 114. Optimized conditions for cloud point extraction were 3% Triton X 114 (w/v), 0.1 M ammonium acetate, and heating at 50°C for 30 min. This approach proved effective in giving quantitative recoveries from a diverse range of food matrixes, and optimized LC gave baseline chromatographic separation for all colors including Sudan IV and Red B. Single-laboratory validation was performed with spiking into liquid matrixes (wine and homemade wine), semiliquid matrixes (sauce and homemade paprika paste), and solid matrixes (spice and homemade chili powder) using the respective blank matrixes for matrix-matched calibration. The LOQ values for water-soluble colors were in the range of 15–150 mg/kg, and for the fat-soluble colors, 0.1–1.5 mg/kg. The mean recovery values were in the range of 69.6–116.0% (except Allura Red and Sunset Yellow in wine, for which recoveries were lower). The mean RSDs for colors were in the range of 4.0–14.8%. A small survey was conducted of samples of confectionery products, dried fruits, wines, bitter sodas, juices, sauces, pastes, and spices, which demonstrated the applicability of the method to a diverse selection of real food samples. Allura Red was detected in strawberry jelly and Sunset Yellow in artificial saffron.

Xiangaing pan et al.34 was worked on effects of twelve commercially available synthetic food colorants, including tartrazine, rose bengal, brilliant blue FCF, new coccin, amaranth, erythrosine B, phloxine B, indigo carmine, acid red, fast green FCF, allura red AC and sunset yellow FCF, on photosensitized oxidation of methyl linoleate (MeLe) were investigated. Rose bengal, erythrosine B and phloxine B accelerated oxidation of MeLe under light exposure and
Chapter 03 Literature Survey
their pro-oxidative effects were concentration-dependent. Light exposure of MeLe with added colorants induced generation of hydroperoxide isomers, including 10-cis,trans- and 12-cis,trans-MeLe hydroperoxide, suggesting that the food colorants served as photosensitizers under the present conditions. The addition of ?-tocopherol or ?-carotene effectively suppressed the oxidation of MeLe, and their antioxidative effects were concentration-dependent. Neither ?-tocopherol nor ?-carotene altered the distributions of hydroperoxide isomers of MeLe. This study showed that the food colorants containing a xanthene skeleton increased their potential as photosensitizers with increase in both the number and atomic mass of halogen substituents on the xanthene skeleton.

A.Husain et al.35 worked on to assess the intake of artificial food color additives by 5–14-year-old children in the State of Kuwait, a 24-h dietary recall was conducted twice on 3141 male and female Kuwaiti and non-Kuwaiti children from 58 schools. The determination of color additives in 344 foods items consumed was performed using high-performance liquid chromatography with diode array detector. A comparison with the Food and Agriculture Organization and World Health Organization acceptable daily intakes (ADIs) was undertaken to evaluate the potential risk associated with the consumption of artificial color additives by children in Kuwait. The results indicated that out of nine permitted colors, four exceeded their ADIs by factors of 2–8: tartrazine, sunset yellow, carmoisine and allura red. Further, follow-up studies to provide insight into potential adverse health effects associated with the high intakes of these artificial color additives on the test population are warranted.

Teresa Fortuna et al.36 the aim of this work was to determine red synthetic dyes in traditional sweets. commercially sold at traditional open Easters market using high performance liquid chromatography. Four red synthetic dyes: azorubine (E122), amaranth (E123), cochineal red (E124), and Allura red (E129) were determined. On the basis of the results it was found that the most commonly used dye in traditional sweets was cochineal red – it occurred in 17 samples. Allura red was used only in one sample, whereas no sample contained azorubine. Six samples of sweets contained two kinds of red dyes. Amaranth and cochineal red contents ranged from 9.8 to 584.8 mg/kg and from 7.3 to 198.4 mg/kg, respectively. The maximum usage level of synthetic dye was exceeded only in one product, however most of the samples were incorrectly labelled,
Chapter 03 Literature Survey
with lack of obligatory information about synthetic dyes used and that the synthetic dyes may have an adverse effect on activity and attention in children.

Mannan Hajimahmoodi et al.37 was worked on eight synthetic food colors were analyzed by green liquid chromatography. Green liquid chromatography is an environmentally friendly technique which does not use organic solvents in the extraction procedure or in the chromatographic method. Analysis was carried out for the following colors: tartrazine (E102), indigo tine (E132), Quinoline Yellow (E104), Ponceau 4R (E124), Sunset Yellow (E110), Brilliant Blue (E133), Allura Red (E129) and carmoisine (E122) in four different foods: cookies, colored rice, and saffron and fruit juice. The method was performed on an Eurospher-100 C8 (5 ?m, 4.6 × 250 mm) column with ultraviolet (UV)-VIS detection and validated by determining the calibration lines, measurement of recovery, precision, and limits of quantification and detection (LODs and LOQs). LOD ranged from 0.04 mg kg–1 for E102 to 1.00 mg kg–1 for E122; LOQ ranged from 0.06 mg kg–1 for E102 to 1.12 mg kg–1 for E122. The levels of colors in foods were compared with Iranian National Standards, but only 7.5% of cookies, 30% of colored rice, 8% of saffron and 12% of juice samples were in compliance with these standards. Tartrazine is prohibited in Iran, but it was found as the most prevalent food color in the samples analyzed. The results of these tests confirmed that HPLC avoiding the use of organic solvents is a suitable method and can be used for quantitative analyses or screening of food samples for synthetic food colors.  
Syed Saeed-ul- Hassan et al. 38 was worked on synthetic food dyes are being used commonly for enhancement of visual appeal, in different candies and bubble gums. To see if permitted food colors are being used in candies and bubble gum or not. At least 40 samples of different candies and bubble gums were collected from the market and dye stuffs used in them were separated by wool extraction method. The dyes were identified by color reaction, paper chromatography and spectrophotometry. It was found that nearly all the samples collected from high to medium standard shops had permitted colors which were within the prescribed limits. However samples which were unwrapped and were collected from low standard shops contained non-permitted colors which are harmful and carcinogenic. Non permitted food colors should not be used in food items as they have a harmful effect on health and are carcinogenic.

Chapter 03 Literature Survey
Irena Baranowska et al.39 was worked on TLC methods have been developed for analysis of food pigments, sweeteners, and a preservative. Patent blue V, quinolone yellow, brilliant blue FCF, tartrazine, azorubine, ponceau 4R, curcumine, indigo carmine, cochineal, methyl violet, mixed carotenes, plain caramel, erythrosine B, and orange yellow S were separated on silica gel G with isopropanol-(12.5%) aqueous ammonia, 10 + 2 ( v/v ), as mobile phase. Aspartame, acesulfame K, sodium cyclamen, and benzoic acid were separated on thin layers of silica gel G with ethanol-isopropanol-(12.5%) aqueous ammonia, 10 + 40 + 1 ( v/v ), as mobile phase. These chromatographic systems were applied to the analysis of food additives in 23 sparkling and non-sparkling drinks.

CHAPTER NO.4
THEORETICAL BACKGROUND
Chapter 04 Theoretical Background
4333875-18161004.1. THIN LAYER CHROMATOGRAPHY
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is
performed on a sheet of glass, plastic, or aluminum foil, which is
41814760Fig 4.1 Thin Layer Chromatography ccccccccccccccccccccccchchCCCCCCChromatography00Fig 4.1 Thin Layer Chromatography ccccccccccccccccccccccchchCCCCCCChromatographycoated with a thin layer of adsorbent material, usually silica gel,
aluminum oxide, or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analysts ascend the TLC plate at different rates, separation is achieved. 40
Thin-layer chromatography can be utilized to screen the advancement of a response, recognize mixes exhibit in a given blend, and decide the virtue of a substance. Particular cases of these applications include: breaking down and unsaturated fats, recognition of pesticides or bug sprays in nourishment and water, examining the color creation of filaments in crime scene investigation, measuring the radiochemical immaculateness of radiopharmaceuticals, or recognizable proof of restorative plants and their constituents. Various upgrades can be made to the first technique to mechanize the diverse strides, to build the goals accomplished with TLC and to permit more precise quantitative examination. This technique is alluded to as HPTLC, or “elite TLC”41
4.1.1 Preparation Of Thin Layer Chromatography Plate
TLC plates are generally commercially available, with standard molecule measure extents to enhance reproducibility. They are set up by blending the adsorbent, for example, silica gel, with a little measure of latent cover like calcium sulfate (gypsum) and water. This blend is spread as a thick slurry on an inert bearer sheet, typically glass, thick aluminum thwart, or plastic. The resultant plate is dried and initiated by warming in a stove for thirty minutes at 110 °C. The thickness of the permeable layer is normally around 0.1 – 0.25 mm for explanatory purposes and around 0.5 – 2.0 mm for preparative TLC 42
Chapter 04 Theoretical Background
1265274top
Fig: 4.2 Steps to prepare TLC plates.

4.1.2. Separation Process
Different compounds in the sample mixture travel at different rates due to the differences in their attraction to the stationary phase, and because of differences in solubility in the solvent. By changing the solvent, or perhaps using a mixture, the separation of components (measured by the Rf value) can be adjusted. Also, the separation achieved with a TLC plate can be used to estimate the separation of a flash chromatography column.43

Fig: 4.3 Development of TLC Plate Separation.

Development of a TLC plate, a purple spot separates into a red and blue spot. Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds that differ in polarity, the more polar compound has a stronger interaction with the silica and is, therefore, more capable to dispel the mobile phase from the binding places.44

Chapter 04 Theoretical Background
As a consequence, the less polar compound moves higher up the plate (resulting in a higher Rf value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate. It is commonly said that “strong” solvents (eluents) push the analyzed compounds up the plate, whereas “weak” eluents barely move them. The order of strength/weakness depends on the coating (stationary phase) of the TLC plate.45
For silica gel coated TLC plates, the eluent strength increases in the following order: Perfluoroalkane (weakest), Hexane, Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl ether, Ethyl acetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water, Methanol, Triethylamine, Acetic acid, Formic acid (strongest).46
4.1.3. Application
Purity of  any Sample : Purity of sample can be carried out with TLC. Direct comparison  is done between the sample and the standard or authentic sample; if any impurity is detected, then it shows extra spots and this can be detected easily.

Identification of Compounds: Thin layer chromatography can be employed in purification, isolation and identification of natural products like volatile oil or essential oil, fixed oil, waxes, terpenes, alkaloids, glycosides, steroids etc.

Examination of Reactions: Reaction mixture can be examined by Thin layer chromatography to access whether the reaction is complete or not. This method is also used in checking other separation processes and purification processes like distillation, molecular distillation etc.47
Limitation
Although it is a very simple and convenient technique, one of its limitations is that it cannot tell the difference between enantiomers and some isomers. Another disadvantage of TLC is that in order to identify specific compounds, the Rf values for the compounds of interest must be known beforehand.48
Chapter 04 Theoretical Background
3731895412750004.2. SPECTROPHOTOMETER
40614602169160Fig4.2 Spectrophotometer
00Fig4.2 Spectrophotometer
Spectrophotometry is a quantitative measurement of the reflection or transmission properties of a material as a function of wavelength. It is more specific than the common term electromagnetic spectroscopy which deals with visible light near ultraviolet and near infra-red. It does not cover the time resolved spectroscopic techniques which means that “anything that allows to measure temporal dynamics and kinetics of photo physical processes”. Spectrophotometry involves the use of
spectrophotometer. 48
A spectrophotometer is an instrument which is used to measure the intensity of electromagnetic radiation at different wavelengths. Important features of spectrophotometers are spectral band width and the range of absorption or reflectance measurement. Spectrophotometers are generally used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids such as polished gases or glass. They can also be designed to measure the diffusivity on any of the listed light ranges in electromagnetic radiation spectrum that usually covers around 200 nm-2500 nm using different controls and calibrations.4849
4.2.1 Principle of Spectrophotometer
Light source, diffraction grating, filter, photo detector, signal processor and display are the various parts of the spectrophotometer. The light source provides all the wavelengths of visible light while also providing wavelengths in ultraviolet and infra-red range. The filters and diffraction grating separate the light into its component wavelengths so that very small range of wavelength can be directed through the sample. The sample compartment permits the entry of no stray light while at the same time without blocking any light from the source. The photo detector converts the amount of light which it had received into a current which is then sent to the signal processor which is the soul of the machine.49
Chapter 04 Theoretical Background

center0Fig 4.5 Principle Of Spectrophotometer
00Fig 4.5 Principle Of Spectrophotometer

4.2.2 Types Of Spectrophotometer
There are 2 major classifications of spectrophotometer. They are single beam and double beam.

A double beam spectrophotometer compares the light  intensity between 2 light paths, one path containing the reference sample and the other the test sample.

A single beam spectrophotometer measures the relative light  intensity of the beam before and after the test sample is introduced.50
Even though, double beam instruments are easier and more stable for comparison measurements, single beam instruments can have a large dynamic range and is also simple to handle and more compact51
Historically, spectrophotometers use a monochromatic containing a diffraction grating to produce the analytical spectrum. The grating can either be movable or fixed. If a single detector, such as a photomultiplier tube or photodiode is used, the grating can be scanned stepwise so that the detector can measure the light intensity at each wavelength (which will correspond to each “step”). Arrays of detectors, such as charge coupled devices (CCD) or photodiode arrays (PDA) can also be used. In such systems, the grating is fixed and the intensity of each wavelength of light is measured by a different detector in the array. Additionally, most modern mid-infrared spectrophotometers use a Fourier transform technique to acquire the spectral information. The technique is called Fourier transform infrared spectroscopy.58Many older spectrophotometers must be calibrated by a procedure known as “zeroing.” The absorbency of a reference substance
Chapter 04 Theoretical Background
is set as a baseline value, so the absorbencies of all other substances are recorded relative to the initial “zeroed” substance. The spectrophotometer then displays % absorbency (the amount of light absorbed relative to the initial substance).52
In short, the sequence of events in a modern spectrophotometer is as follows:
1.The light source is imaged upon the sample.

2.A fraction of the light is transmitted or reflected from the sample.

3.The light from the sample is imaged upon the entrance slit of the monochromatic.

4.The monochromatic separates the wavelengths of light and focuses each of them onto the photo detector sequentially 53.

4.2.3. Application
Estimating dissolved organic carbon concentration.Specific Ultraviolet Absorption for metric of aromaticity. The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, and molecular biology. They are widely used in many industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well in laboratories for the study of chemical substances. Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what substances are present in a target and exactly how much through calculations of observed wavelengths54
4.2.4. Limitation
In general, a UV-Visible spectrophotometer(the type you more than likely used) is superb for quantitative analysis but very poor for qualitative analysis. A graph of absorbance vs. wavelength will generally reveal one big, broad peak which will tell you a little bit about the nature of the double bonds(if present), however this is about the extent of the qualitative information.55
A UV-visible spectrophotometer can only be used if the substance under analysis has significant absorbance in the UV or visible range. Not all compounds have this. 
Chapter 04 Theoretical Background
Sometimes, other molecules present in the sample can also absorb at the wavelength under consideration. This can result in an incorrectly high concentration value.56
CHAPTER NO. 5
EXPERIMENTATION
Chapter 05 Experimentation
5.1. SAMPLE COLLECTION:
Confectionaries like custard, jellies, and beverages were collected from Hyperstar Lahore Pakistan in summer 2014 and stored in brown paper at room temperature in dark place prior to analysis.

Table 5.1 List Of Collected Confectioneries Samples
Sr. # Product Name Sample Code Manufactured By
1 Custard vanilla A Delve Desert
2 Custard mango B Delve Desert
3 Custard strawberry C Delve Desert
4 Jelly orange D Delve Desert
5 Jelly mango E Delve Desert
6 Jelly crystals F National
7 Open red jellies G Not branded
8 Tang mango H Kraft foods
9 Tang strawberry I Mondelez
10 Garden falsa J Quershi
11 Tang freeze(watermelon + strawberry) K Mondelez
12 Vimto (sparkling fruit flavor drink) L Nichols plc
13 Sting berry blast M PepsiCo
14 Gatorade grade N  PepsiCo 
15 Gourmet sherbet e jam O Gourmet
16 Jam-e-sherin P Quershi
17 Non branded Q Not branded
Chapter 05 Experimentation
5.2. Sample Preparation
Chemicals
5 % Acetic acid
Silica gel 60 GF254 for TLC
Distilled Water
Propanol (Manufactured by MERCK)
35 % NH3 (BDH Laboratories Supplies)
Apparatus
Measuring cylinder (25 ml,100ml)
Beakers (50 mL,100ml)
Test tubes
Water bath
Glass Stirrer
Bunsen burner
Chromatographic tank
Procedure
As food colorants are added to foods at low concentrations therefore it is necessary to extract and concentrate the colorants to obtain sufficient amount for analysis .Extraction of food dyes was done using standard method AOAC named as Wool Extraction Method. 57
15 cm strips of woolen yarn were taken for each sample. To remove the fluorescent dyes from the yarn, each strip was placed into a different large test tubes with enough household vinegar (5% acetic acid) was used to cover it. Strips should be fully dipped into the vinegar. Tubes were placed into the beaker of boiling water for 5 minutes.

Test tubes were removed from water bath by using a stirring rod, strips of yarn were fished out and the strips were placed on blotting papers. When the test tubes were cooled enough to touch, the vinegar were discarded, the test tubes were rinsed with tap
Chapter 05 Experimentation
water and then with distilled water. custard powder and, jellies powder were poured separately into each test tube and then enough vinegar was poured into each test tube just to cover the sample.

Test tube was hold with holder and dipped it into boiling water to heat the vinegar and to dissolve the color uniformly. The color came out very fast, usually within a few seconds, so it is usually not necessary to kept it in there for longer than 10 seconds. Immediately the colored solution was decanted into a clean test tube. The experiment was repeated with the remaining samples in the same manner.

To extract the dye from the solution on to the strips of yarn, one piece of yarn was placed into each tube of the colored solution and 4mL of 5% acetic acid was added in all tubes. test tube was heated on boiling water bath for 5 minutes. Tubes were removed when yarn took the color of dye. dyed yarn was removed from the solution and rinsed with distilled water.

To extract the dye from the yarn it was brought back into solution, large sized test tubes were taken and one yarn piece was placed into each tube and 3mL of 1M ammonia was added into it. It is essential to use stirring rod for mixing and immersing the yarn.the tube containing yarn and ammonia was heated in the water bath for 5 minute or until color of yarn was faded. yarn was removed as color from the yarn extracted.

To concentrate the dye, dye was placed in a beaker and carefully heated on Bunsen burner. much care was taken so that the dye could not got dry otherwise heat from the flame would decompose the dye. When concentrated, return the dye to a clean small test tube and covered it.

Food dye after extraction from confectionaries (samples) were analysed by chromatographic analysis, spectroscopic analysis respectively.

Chapter 05 Experimentation
5.3. CHROMATOGRAPHIC ANALYSIS
Chromatographic analysis including thin layer chromatography was carried out to separate mixture of dyes and to identify the individual dye components
5.3.1. Thin Layer Chromatography
Separation and identification was accomplished by thin layer chromatography (TLC). To run a thin layer chromatography, the following procedure is carried out:
A small spot of color extracted from sample A is applied to a plate, about 1.5 centimeters from the bottom edge. The solvent is allowed to completely evaporate off, otherwise a very poor or no separation will be achieved. If a non-volatile solvent was used to apply the sample, the plate needs to be dried in a vacuum chamber.

A small amount of an appropriate solvent (eluent) is poured into a glass beaker or any other suitable transparent container (separation chamber) to a depth of less than 1 centimeter. A strip of filter paper is put into the chamber so that its bottom touches the solvent and the paper lies on the chamber wall and reaches almost to the top of the container. The container is closed with a cover glass or any other lid and is left for a few minutes to let the solvent vapors ascend the filter paper and saturate the air in the chamber. (Failure to saturate the chamber will result in poor separation and non-reproducible results).

The TLC plate is then placed in the chamber so that the spot(s) of the sample do not touch the surface of the eluent in the chamber, and the lid is closed. The solvent moves up the plate by capillary action, meets the sample spot and carries it up the plate (elutes the sample). The plate should be removed from the chamber before the solvent front reaches the top of the stationary phase (continuation of the elution will give a misleading result) and dried.

The same procedure was carried out for the remaining samples from B to M.

Chapter 05 Experimentation
Their Rf value is calculated by using following formula
Rf        =      Distance travelled by the dye
Distance travelled by the solvent front
Where Rf is the retardation factor.

5.3.2. Spectrophotometer
Uv- visible spectrophotometer was used to determine the lambda max of the samples color. Results were computed and discussed in the following chapter.

CHAPTER NO.6
RESULTS ; DISCUSSION
Chapter 06 Result and Discussion
6.1. COMPARISON BETWEEN FOOD COLOURS STATED ON THE LABEL ; IDENTIFIED BY TLC
Table 6.1 showed comparison of food color labeled on the packing and identified by thin layer chromatography.

Sample A, B, D, and E contain allura red and tartrazine. Result confirmed the presence of both colors. Sample H contained sunset yellow and allura red .Result showed that both colors are present. Sample H, I, J, K, L, M, N, O and P contained allura red and results showed the presence of allura red. Sample G, and Q contained red colors but result showed these samples did not contain allura red AC.

Table 6.1:Comparison b/w food colors stated on the label ; identified by TLC.

Sr. # Sample Food color stated on label Food color identified by TLC
1 A Tartrazine , Allura red Tartrazine, Allura red
2 B Tartrazine, Allura red Tartrazine, Allura red
3 C Allura red Allura red
4 D Tartrazine, Allura red Tartrazine, Allura red
5 E Tartrazine, Allura red Tartrazine, Allura red
6 F Allura red Allura red
7 G NIL NIL
8 H Sunset yellow
Allura red Sunsetyellow,Allura red
9 I Allura red Allura red
10 J Allura red Allura red
11 K Allura red Allura red
12 L Allura red Allura red
13 M Allura red Allura red
Chapter 06 Result and Discussion
14 N Allura red Allura red
15 O Allura red Allura red
16 P Allura red Allura red
17 Q NIL NIL
6.2. Thin Layer Chromatograms
Fig 6.1 to 6.17 showed that thin layer chromatograms
446532039179500

4252236135890Fig.6.3: Sample C
0Fig.6.3: Sample C
1957981106680Fig.6.2: Sample B
0Fig.6.2: Sample B
-88900101600Fig 6.1: Sampal A00Fig 6.1: Sampal A
Chapter 06 Result and Discussion
39655752765425Fig. 6.6: Sample F
00Fig. 6.6: Sample F

-13208041910Fig.6.4: Sample D
00Fig.6.4: Sample D
199453543180Fig.6.5: Sample E
00Fig.6.5: Sample E

213423538100Fig 6.8: Sample H
00Fig 6.8: Sample H
42487859525Fig 6.9: Sample I
00Fig 6.9: Sample I
-1333499525Fig 6.7: Sample G
00Fig 6.7: Sample G

Chapter 06 1987554053840Fig 6.10: Sample J
00Fig 6.10: Sample J
Result and Discussion
42672003740150026543041910000-39839902795905Fig 6.8: Sample K
00Fig 6.8: Sample K

586105317500026536653045460Fig 6.12: Sample L
00Fig 6.12: Sample L
4914903045460Fig 6.11: Sample K
00Fig 6.11: Sample K

19050039116000
2933700115570006083304889500-14141452948305Fig. 6.13: Sample M
0Fig. 6.13: Sample M
5162552948305Fig.6.14: Sample N
0Fig.6.14: Sample N
28663902948305Fig 6.15: Sample O
00Fig 6.15: Sample O

Chapter 06 Result and Discussion
24485602743835Fig 6.17: Sample Q
00Fig 6.17: Sample Q
-469902753360Fig 6.16: Sample P
00Fig 6.16: Sample P

6.3. Rf ; ?max Of Thin Layer Chromatograms
Table 6.2 showed Rf and ?max of samples from A to Q.

Table 6.2 Rf value and ?max(nm) of Samples A to Q
Sr.# SAMPLES Rf VALUES ?max(nm)
Allura Red Dye 0.51-0.60 504
1 A 0.59 505nm
2 B 0.57 504nm
3 C 0.62 503nm
4 D 0.56 504nm
5 E 0.53 504nm
6 F 0.57 504nm
7 G Not standard Not standard
8 H 0.56 504nm
9 I 0.56 504nm
10 J 0.62 503nm
11 K 0.60 504nm
12 L 0.52 504nm
13 M 0.56 505nm
14 N 0.57 504nm
15 O 0.60 504nm
16 P 0.52 503nm
17 Q Not standard Not standard
Chapter 06 Result and Discussion
The standard reference values of Rf and ?-max for allura red AC are; Rf = 0.51-0.60, ?-max = 504nm 58
TLC results shows that Rf values for sample A,B,D,E,F,H,I,K,L,M,N,O ; P lies in the standard reference range. UV results also meet the standard reference value. It means these food products contain permitted synthetic food color allura red. However, TLC results for sample C and J are slightly different form the standard reference value. But their ?max values lies in standard range. This variation in Rf value may be due to complex matrix.

The Rf and ?-max value for sample G ; Q does not lie in the standard reference range, either their values are too much high or too much low when compared to standard reference values. It means that in both of these samples non permitted or sub-standard food colors are present and allura red is absent.

4612005-209600800Synthetic colors are also harmful if they are not used in prescribed limits. But non-permitted colors are lethal for human health if they are added in eatables. That’s why, sub-standard colors should not be used.

CHAPTER NO.7
SUMMARY & CONCLUSION
Chapter 07 Summary and Conclusion
Various samples of custards, jellies, beverages were collected from variety of shops having different components of food dyes. Data collected from extraction of dye and analyzed by chromatography and spectroscopy confirmed that reference values of dyes from samples fell in the range of 0.51 – 0.60 indicating that all the samples contained allura red as food dye. Although synthetic dyes added in the food are selected on the basis of minimum risk but the degradation products of certain dyes can pose serious health hazard and could cause diseases like cancer, brain tumor, allergy, chromosomal damage,
A thin Layer Chromatography method has been developed to identify the occurrence of food coloring. we use Thin Layer Chromatography for identification of allura red in samples from A to Q due to following reasons Less equipment is required. Very little time for separation is required, It is more sensitive, The lower detection limit of analytical sample in TLC is approximately one decimal lower than that is paper chromatography and very small quantities of sample is sufficient for analysis, Spraying with corrosive agents for identification is also permitted which is not possible in paper chromatography as cellulose gets destroyed, This method is used for adsorption, partition, ion exchange chromatography as there is wide range of adsorbents available, The individual samples do not get diffused as compared to paper chromatography hence sensitivity of detection is more, The components which are separated can be recovered easily by scratching the powdery coating of plate and quantitative separation of spots or zone are possible, It is possible to visualize the components for identification by UV light as the inorganic adsorbent background does not fluoresce. This method can be applied to preparative separation with the help of thicker layers of adsorbents.

The FDA argues that food coloring is safe because it causes cancer only when injected into rats, not when they are feeding on it. But this prove wrong because all certified food colors in use today belong to polycyclic aromatic hydrocarbon class are universally suspected as carcinogenic. It is therefore felt, that use of dyes which are added merely to enhance the aesthetic appeal without any food value, should either be banned and consumers particularly children must be educated about the possible health hazards of the colored food they consume. Non permitted food colors should not be used in food items as they have a harmful effect on health and are carcinogenic.

Chapter 07 Summary and Conclusion
Suggestion
Non permitted food colors should not be used in food items as they have a harmful effect on health and are carcinogenic.

There should be quality check on food colors.

Thin layer chromatography is an efficient method for identification of food colors. It should be used by industrial or non-industrial sectors.

CHAPTER NO.8
REFERENCES

Chapter 08 References
REFERENCE
1. Fairchild. E ,Alyssa,H.,Andrea,B., Carlo, P.& Roberrrrtai, P., ” A Source Book of the Toxic Effects of Chemical substances” (1978),189-195.

2.     Al- Degs, Abebe,M.,Weiping,L.,Wendy,S.,Bussie,M.&Torbert,R, “Determination of Three Dyes in Commercial Softdrinks using HLA/GO Liquid Chromatography” (2009),302-303.

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