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By Pnotporn Jantarakolica (u5861115) Pichayathida Siriwechdaruk (u5861105)
Supervised by Dr.Manchuta Dangkulwanich and Ms.Natamon Rittilertnapa

A scientific technique, Thin Layer Chromatography (TLC) along with quantitative determination through the usage of digital processing of
images obtained by thin-layer chromatography has been used to identify the type and quantify the artificial food coloring contained within
the tea leaves. Six brands of commercial Thai tea leaves (Seven types of tea leaves) were tested and the dyes that are being identified are:
Tartrazine, Ponceau 4R, Carmoisine, and Sunset Yellow. The result obtained shows that all of the samples contain at least one type of
artificial food coloring; Sunset Yellow, and six out of seven tea leaves brands tested are proven to be falsely labelled, though; the quantity of
the food color additives complies with the ADI set by JECFA and the Scientific Committee on Food of the European Commission (SCF).

Are there any artificial coloring incorporated in Thai teas?
If Thai teas were being tested by the technique of Thin Layer Chromatography, would there be any artificial coloring detected?

If commercial Thai tea leaves were testified in order to find artificial food colorings through Thin Layer
Chromatography technique, then several types of artificial food coloring would be identified.

Literature Review
From the testification conducted by Institution of Nutrition, Mahidol University, the majority of commercial Thai Tea
leaves contain artificial coloring, namely, Tartrazine, Ponceau 4R, Carmoisine, and Sunset Yellow (SMBUYER Magazine
Editorial team, n.d). Though the Thai Food and Drug Administration allows industries to incorporate food coloring
additives into their food products under the limit of 70 mg per 1 kg of food product (or 50 mg per 1 kg of food product for
Ponceau 4R), artificial coloring is banned according to the 196th issue of the announcement from the administration
(Dabbaransi, 2000).
As of the artificial food colorings reportedly found within tea leaves, there are several researches that has proven the
harm of consumption. Moreover, several countries had banned industries from adding the stated dyes into their food
products. Sunset yellow FCF (FD&C Yellow 6 or E110) has been banned in Finland and Norway. It has been claimed that
sunset yellow may result in the consumer having these following effects: gastric upset, diarrhea, vomiting, nettle rash
(urticaria), and swelling of skin (angioedema). The ADI according to the JECFA and the Scientific Committee on Food of
the European Commission (SCF) (acceptable daily intake amount) of Sunset Yellow is 2.5 mg/kg bw/day (König,2015).
In contrast to Sunset yellow FCF, the recommended amount of daily consumption of tartrazine (FD&C Yellow 5 or E102)
is 7.5mg per kg of body weight, but only 5% of it will be absorbed in a human body and overconsumption may cause
harm. It has been found as an activator of oestrogen receptor and the cause of biliary cirrhosis in postmenopausal
women. In addition, it also binds to bovine serum albumin, forming a complex, abrupting physiological functions. It
has been banned in Norway and Austria due to its effect in allergies, asthma, skin rashes, hyperactivity, and migraines
(Ramesh & Muthuraman, 2018). Ponceau 4R (Ext D&C Red 8 or E124) is permitted in food with the recommended ADI
values by Food and Agriculture Organization (FAO) and World Health Organization (WHO) of 0-4.0 mg/kg body weight.
However, The US Food and drug administration has banned the use of Ponceau 4R in food and declare it as Ext D&C
red 8, which means it is only approved for the usage in the external drug and cosmetic industry. Furthermore, China
has a strict restriction on the maximum amount of ponceau 4R authorized in food products established by the Ministry
of Health (Wang, Gao, Sun, & Zhao). Other than the US, the food coloring is banned in Canada, Denmark, Sweden,
Norway and Japan due to its carcinogenic and mutagenic effects (Xie et al., 2012; Izzat, 2017). Similarly to ponceau 4R,
carmoisine (Azorubine, Ext D&C Red #10, E122) is suspected in its carcinogenic and mutagenic effects and is allowed
for external drugs and cosmetics only in the United States. Apart from that, various of researches had observed that it
is likely that carmoisine may lead to negative effects on the liver. It is banned as food additive in the United States,
Canada, Japan, Norway, and Sweden (Siva, 2014; Izzat, 2017). The ADI of carmoisine is 4 mg/kg bw (König,2015).
Regulation EC 1333/2008 EU has instructed that food dyes such as Tartrazine, Ponceau 4R, Carmoisine, and Sunset
Yellow must be labeled with E number of color to emphasize that they may cause harm to children and has eliminated
the use of some of these colors in their country. (McAvoy, 2014) Despite this, in 1970, 9.5% of United States children has
been affected with attention deficit hyperactivity disorder (ADHD), leading to an assumption that these food additives
may be its causes (Ramesh & Muthuraman, 2018). The controversy revolving around the relationship between artificial
food colorings and ADHD has been discussed for more than 35 years long, with accumulating evidences from imperfect
studies. However, many scientists have agreed that ADHD might be a contributing factor of ADHD and the fact that the
per capita daily consumption of artificial food colorings have been continuously increasing in the past could lead to
worse health risks than ADHD (Arnold, Lofthouse & Hurt, 2012).
According to “A review of chromatographic methods for determination of synthetic food dyes” written by Kucharska &
Grabka (2009), there are many methods that can be used to separate the dyes such as thin-layer chromatography (TLC),
spectrometry, adsorptive voltammetry, differential pulse polarography, capillary electrophoresis, and high performance
liquid chromatography (HPLC) and ion chromatography. However, due to the limited time and resources, the method to
identify the artificial food coloring in tea leaves is limited to TLC, which is the most simple and common way of dye
separation through the usage of difference in polarity. The very first stage before conducting TLC is to separate sugars,
fats, and other substances so that the dyes will be clearly seen after running the TLC. By this, the most efficient way that
has been mentioned is boiling acetic acid solutions of the samples with white wools. To remove impurities, water will be
used to wash the wool. Then after boiling it with ammonia, the dyes will eventually come off from the fabric. After that,
wait for it to evaporate. However, in the conducted experiment, this process has been neglect because it was
unnecessary with Thai tea as most of the sample does not contain sugar and although one of the samples contains
vanilla extract, the extract doesn’t affect the overall result. The mechanism behind TLC is the difference in polarity. TLC
would provide spatial separation of the food colorings and have the ability make simultaneous separation. For
quantitative determination, a scanner is used at the optical resolution of 300 dpi. Softwares such as Macherey-Nagel
TLC Scanner software could be used as suggested by the paper written by Soponar, Moţ, and Sârbu (2008) or use the
program ColorDens as suggested by Gerasimov (2003). However, the software couldn’t be obtained. Hence, another
software called “ImageJ” which is usually used in gel electrophoresis was used instead as the program could also
determine the intensity of the band and could give out a regression linear equation as well when the data is plotted
into a graph.

Arnold, L. E., Lofthouse, N. & Hurt, E. (2012). Artificial Food Colors and Attention-Deficit/Hyperactivity Syptoms: Conclusions to Dye for. Retreived 18 February, 2018 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441937/
Dabbaransi, K. (2000). The 196th announcement from the Food and Drug Administration : Teas. Retrieved 17 February, 2018. From http://taxclinic.mof.go.th/pdf/417D2E7B_DDE9_AC5E_A773_B5F37A9733E8.pdf
Gerasimov, A. V. (2003). Use of Software Processing of Standard Chromatogram Images in Quantitative Planar Chromatography. Retrieved 18 February, 2018 from
Izzat, S. S. (2017). A Surveillance study on the presence and safety of different types of food additive in children’s foods available in the markets. Retrieved 18 February, 2018 from http://jzs.univsul.edu.iq/issues/vol19n1/jzs-10580
Kucharska, M. & Grabka, J. (2009). A review of chromatographic methods for determination of synthetic food dyes. Retrieved February 18, 2018 from https://www.sciencedirect.com/science/article/pii/S0039914009007395
König, J. (2015). Food colour additives of synthetic origin. Retrieved 18 February, 2018 from https://www.sciencedirect.com/science/article/pii/B9781782420118000027
McAvoy, S. A. (2014). Global Regulations of Food Colors. Retrieved 18 February, 2018 from https://www.iacmcolor.org/wordpress/wp-content/uploads/2014/09/PMCA-2014-McAvoy.pdf
Naik, P. (2014). Food colourants and health issues: are we aware?. Current Science, 106 (2). Retrieved from http://www.jstor.org/stable/i24099727
SMBUYER Magazine Editorial Staffs. (n.d.). ชาโบราณยี่ห้อไหนใส่สีมากเกินไป. นิตยาสารฉลาดซื้อ,110 Retrieved from https://www.chaladsue.com/article/470/อาหาร-ฉบับที่-110-ชาโบราณยี่ห้อไหนใส่สีมากเกินไป
Soponarm F., Moţ, A. C., & Sârbu, C. (2008). Quantitative determination of some food dyes using digital processing of images obtained by thin-layer chromatography. Retrieved 18 February, 2018 from
Wang, M., Gao, Y., Sun, Q. & Zhao, J. (2014). Ultrasensitive and simultaneous determination of the isomers of Amaranth and Ponceau 4R in food based on new carbon nanotube/polypyrrole composites. Retrieved 18 February, 2018
from https://www.sciencedirect.com/science/article/pii/S0308814614015465
Xie, Y., Li, Y., Sun, Y., Wang, H., Qian, H., & Yao, W., (2012). Theoretical calculation (DFT), Raman and surface-enhanced Raman scattering (SERS) study of Ponceau 4R. Retrieved 18 February, 2018 from

Mr. Nattapat Poolyam, Ms. Sorasaree Tonsiengsom, Mr. Rawinsiwat Dechaanuntasub, Ms. Salinee Thamthavornvanich,
& Mr. Andrew James Filipczak