In this slide contains Methods of Detection of Natural, Permitted and Non Permitted Dyes. Presented by: P.SUDHEER KUMAR (Department of pharmaceutical analysis). RIPER, anantapur

1. RIPER
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement for I year M. Pharm I semester
Presented by
P. Sudheer Kumar (20L81S0712)
M. Pharmacy
Department of Pharmaceutical Analysis
Under the Guidance of
Dr. K.Vinod kumar, M. Pharm., Ph. D
Professor of Pharmaceutical Analysis,
Head of department - Pharmaceutical Analysis.
Method Of Detection Of Natural,
Permitted And Non-permitted Dyes

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
• Introduction
• Properties
• Detection of dyes and pigments
• Detection of chlorophyll in spinach
• Identification of non-permitted food colours
• Thin layer chromatography
• Procedure
• References
Contents

3. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
Colours added to food ingredients has always fascinated human
beings. It is a organoleptic property that can make food attractive,
appealing, appetizing and informative to be acceptable immediately
by the consumer, even before tasting it.
Introduction

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
Below are some reasons why ingredients are added to foods:
 To restore the original exterior appearance of food whose color
changed as a result of processing, storing, packaging and distribution.
 Offsetting color loss due to light, air, extremes of temperature,
moisture.
 May keep certain foods tasting fresher for long time.
 For improving the visual food attractiveness
 To restore the original color.
 Providing identity to foods.
 Decorative and artistic purposes.
 To make food more desirable.
 To mask defects.
WHY FOOD PRODUCTS NEED TO BE COLOURED?

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
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.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
1. Colours:
Properties
NATURAL PIGMENTS COLOURS
Saffron Yellow orange powder
Curcuminoid Yellow
Lycopene Red
Anthocyanins Red to purple to blue
Betalain Yellow and Red
Caramel Brown
Carminic acid Orange to Red
Beta Carotene Orange
Annato Yellow and Orange colour
Chlorophyll Green

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
 Water soluble pigments:
The pigments which are water soluble are basically used to colour
water-based products like brightly coloured soft drinks and jelly.
e.g. Anthocyanins, Betalain, Caramel, Carminic acid, Saffron
 Water insoluble pigments:
The pigments which are soluble in fat are a great choice for
colouring dairy products, which typically have a high fat content like
milk, cheese, margarine.
e.g. Carotenoids, Annatto, Chlorophyll, Curcuminoid
2. Solubility

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Chemical Methods:
1. Detection Of Carotene:
The sample is first dissolved in
chloroform.
 To it add a solution of
antimony trichloride.
 A dark blue color is formed
which indicates the presence of
carotene.
Detection of dyes and pigments

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
2. Detection of Chlorophyll
Chlorophyll sample is first
extracted with ether.
 The ether extract is then treated
with 10% KOH in ethanol.
 The color turns to brown and
then quickly returning to green
indicating that the sample
contains chlorophyll.

10. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
3. Detection Of Caramel:
It can be done by fischer’s
reaction.
 Firstly the sample is extracted
with 50ml ether.
 Heat it in a porcelain dish for
evaporation.
 To the residue add 3 drops of 1%
resorcinol in HCl.
 Appearance of rose color
indicates the presence of caramel
pigment.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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4. Detection of Turmeric
 Firstly extract the sample with
alcohol.
 Then evaporate the alcoholic
extract to dryness.
 Moisten with boric acid and add
few drops of HCl.
 Dry the above solution again.
 If the turmeric is present, the
colour changes from cherry red
to bluish green on the addition
of NaOH.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
5. Detection of Annato
• Shake the melted fat or oil with
2% NaOH solution and pour the
aqueous extract on moistened
filter paper.
• The filter paper will show a straw
colour which will remain with a
gentle wash with water.
• Dry the paper and add a drop of
40% stannous chloride and dry
carefully.
• If the colour turns purple, the
presence of annato is confirmed.

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6. Detection of Cochnineal
The cochineal sample is first treated
with amyl alcohol solution.
Shake an amyl alcohol solution of
the material with dilute ammonia.
 Purple color is produced indicating
the presence of cochineal.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
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Detection of Chlorophyll in Spinach
• Chlorophyll is the green pigment required for photosynthesis in
plants and seaweed.
• Chlorophyll is divided into types a to e, depending on its structure.
• Here, an analysis of chlorophyll a and b in spinach is done.
• Generally, chlorophyll shows a spectrum with characteristic
absorption maximum at 430 to 450 and 650nm regions.
• The detection and spectrum is provided by a photodiode array UV-
Vis detector.

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
Pretreatment
• Add 10ml of acetone to 10g of spinach, and mix.
• Centrifugal separation.(3000rpm for 10min)
• Inject 10µL of the supernatant liquid.
Analytical Conditions
• Instrument : HPLC
• Column : STR ODS-II
• Mobile phase : Methanol
• Temperature : 40 degree celsius
• Flow rate :1mL/min
• Detection : Photodiode array detection from 220nm to 700nm

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Spectrum of Chlorophyll

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Spectrum of Chlorophyll

18. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
• The whole, dried or fresh turmeric is typically free of contamination,
turmeric powder can be adulterated with different chemical powders
which are used as substitutes for curcumin.
• Curcuma zedoaria, a wild relative of turmeric is reported to have mixed
into turmeric due to its close resemblance with it.
• Similarly, Metanil yellow is a toxic dye that has been added to turmeric
powder to mimic the appearance of curcumin when actual content is low.
• Metanil yellow is azo dye synthesized from the coupling of metanilic acid
and diphenylamine.
• Metanil yellow is added in spices only to enhance the colour of spices,
their redness, and they are continuously harming the human health.
Identification Of Non-permitted Food Colours (Metanil
Yellow And Aniline Dyes) In Turmeric Powder

19. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
• Metanil yellow is banned dye as per PFA Act (1954) by Govt. of India, because
same dye has found to be carcinogenic in humans.
• Metanil yellow, the most frequently used non-permitted dye cause insufficient
oxygen supply to skin and mucous membranes along with degenerative
changes in the stomach, kidney, liver and abdomen. It cause cyanosis.
Rapid Test
• Rapid Color Tests are based on specific color changes in the presence of
reagents such concentrated hydrochloric acid and concentrated sulphuric acid.
• These tests get their name from the fact that the color change is observed
within 2-3 minutes on adding the specific reagent.
• The following table shows the different color tests carried out to test the
presence of metanil yellow and aniline dyes in turmeric samples and the
associated color changes.
Contd…

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Contd…

21. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
Thin Layer Chromatography is a chromatographic technique that is used to separate
mixtures containing volatile elements. Thin layer chromatography is used to
determine the presence of adulterant (metanil yellow) in the turmeric samples by
comparing their Rf (ratio of the distance travelled by the sample to the distance
travelled by the solvent) with the Rf of the standard.
Preparation of turmeric samples
• 5 grams of each turmeric sample were taken in a test tube and dissolved in 20mL of
chloroform.
• These were kept overnight in the rotating shaker.
• After 18 hours, these test tubes were removed from the shaker and filtered.
• The filtrate obtained was used for thin layer chromatography.
Thin Layer Chromatography

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Preparation Of Standard Solution
• Stock solutions of the standard (metanil yellow) were prepared by
dissolving 2mg of the standard in 50mL of isopropyl alcohol (solvent).
• This was stored at 10°C.
• The working standard solution was obtained from the stock solution by
diluting it with the solvent (1:5) at the time of analysis.
Preparation of TLC Plates
• Silica gel slurry was prepared in distilled water and then applying it on
the TLC plates (15cm x 7cm) as a thin layer having a thickness of
0.25mm.
• These plates were then allowed to dry.
• These were activated by keeping them at 100C in the oven for 1 hour.

23. RIPER
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
• The samples and standards were spotted onto the TLC plates with the
help of capillaries.
• The solvent used for the development of the TLC plates was a
solution of ethyl acetate, methanol, ammonia, and distilled water
which were mixed in the ratio of 35: 11: 5: 5 respectively.
• These plates were then allowed to develop in a TLC jar for 1 hour
using the above mentioned solvent system.
• The chromatograms obtained were then evaluated under normal light
and the distances travelled by the solvent were measured using a
template scale.
Procedure

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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24
• Their Rf values were then calculated and compared to that of the
standards.
• The results obtained for the given turmeric samples after thin layer
chromatography were expressed in terms of their Rf (retardation
factors) values.
• The Rf values of the standard (metanil yellow) are also calculated in
order to compare and determine the presence of the adulterant; i.e.
metanil yellow in the turmeric samples
Contd…

25. RIPER
AUTONOMOUS
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 25

26. RIPER
AUTONOMOUS
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SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 26
1. Manual of method of analysis of foods, food additives, fssai. 2015;
2. Mandeep Kaur Purba. Detection of Non-permitted Food Colours in
Edibles. Journal of Forensic Research. 2015; 1000S4-003: 2157-
7145.
3. Bhakti Petigara Harp. Qualitative Identification of Permitted and
Non-permitted Colour Additives in Food Products. 2012; 29(6):
886- 896.
References

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NAAC &
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Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 27