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Capstone Project 
Internship at Science Division, Mahidol University International College: 
Qualitative and Quantitative Determination of Artificial Food Coloring In Thai Tea Leaves 
Pnotporn Jantarakolica (5861115) 
Pichayathida Siriwechdaruk (5861105) 
Capstone Mentor: Ms.Natamon Rittilertnapa 
Capstone Teachers: Mr.Andrew J. Filipczak and Mr.Dawit Thepchatree 
Mahidol University International Demonstration School 
Academic Year 2017-18 
The capstone project we decided to do is an internship at Mahidol University International 
College (MUIC), Science Division in the field of Food Science, under the supervision of Dr.Manchuta 
Dangkulwanich. The internship includes working on the identifying and quantifying the artificial 
food coloring additives in Thai tea leaves through the process of Thin Layer Chromatography (TLC).  
Upon the discussion between the members and the advisor at MUIC, we have decided to 
identify the amount of artificial food colorings within the Thai tea since there was a pre-existing 
research done by the institution of nutrition, Mahidol University and the result of the research was 
published through an online magazine called ‘chaladsue’; however, the research wasn’t well known and 
it was conducted eight years ago. Hence, it was decided that an experiment that would determine the 
artificial food coloring in Thai teas would be done in order to prove whether if the companies add 
food coloring to their tea leaves or not and to see whether if there is any changes on the type and the 
amount of dyes added in Thai tea leaves.  
The first three days of the internship (15/9, 21/9, and 29/9) was used to discuss about the 
method that would be used in the experiment according to the pre-existing researches and what should 
be done in order to achieve the goal, which is to be able to quantify and qualify the artificial food 
coloring in Thai tea leaves successfully. At first, three methods were brought to attention: Thin Layer 
Chromatography, Column Chromatography, and High Performance Liquid Chromatography. After 
conducting researches and making discussions, the method of Thin Layer Chromatography was 
chosen as the first process to be done due to the easy access of materials and its effectiveness. After 
Thin Layer Chromatography was completed, High Performance Liquid Chromatography was 
planned to be the second method to do in order to have access to more accurate result.  
After the method was decided, we started the experiment. However, on the first day (9/10) of 
the experiment, there was no standard dyes yet. Hence, the day was spent on discussing the 
information obtained from previous researches about the mobile phase that will be used to separate 
the dyes and deciding on the concentration of the tea solution that would be used. After the discussion 
is finished, by using the mobile phase 10 : 1 : 1 Isopropyl alcohol : Ammonia : Distilled Water the tea 
samples were tested through the process of Thin Layer Chromatography. From the technique, it could 
be observed that all of the tea samples contain at least one common dye and some of the tea samples 
contains more than one type of food dye. Also, it seems like 1 microliter per spot creates too large spot 
on the TLC plate.  
During the second day (25/10), we have worked on the the thin layer chromatography process 
in the lab. For the second lab, the standard dye was acquired and four different mobile phases were 
being tested with different dilution of standard dye mix in order to determine the best one to be used 
to separate the dyes. The four mobile phases are:  
1. Ethyl Acetate : Ethanol: Distilled water (75 : 35 : 30)  
2. Ethyl Acetate : Ethanol : Ammonia (75 : 45 : 15)  
3. Ethyl Acetate : Ethanol : Distilled water (75 : 35 : 30)    
4. Isopropyl Alcohol : Distilled water : Ammonia 30% (10 : 1 : 1)  
The four dilution of standard mix tested were: 25%, 50%, 75%, and 100% 
After the best mobile phase was determined, the tea samples and the standard dye mix were 
tested by using TLC. The spot size was now reduced to 0.6 microliters and the tool used to spot the 
sample was changed from micropipette to microsyringe. From the second experiment, the best mobile 
phase, dilution, and the number and type of dyes within each of the tea sample were acquired. After 
this, several other labs for TLC are needed in order to acquire more accurate information. After the 
current step, which is the qualitative determination using TLC, is finished, I will resume with 
quantitative determination using TLC and computer programs. As of now, there is no changes to the 
plan of the project.  
By conducting the experiment outside of our own school, we have grew a lot as a person. The 
small details we learned throughout the internship such as using pen to collect data instead of pencil 
and do not correct any of the information noted down or labelling every glasswares used for different 
chemicals should not be neglect. We would also label the date since these chemicals do evaporate and 
they may not be good to use if they are kept for too long. Simple things like cutting the plate should 
also be done as straight as we possibly can because it does affect the data. We have also learn how to 
weigh the tea correctly. For instance, using the same balance throughout the experiment and tearing 
every beaker before we place the tea on. Even though each beaker may look the same, but it does not 
necessary mean that they would weight the same. We have also learned that in order to measure the 
distilled water to boil the tea, we should use the graduated cylinder instead of the beaker because they 
are more accurate. Another method in measuring the substance that we have never learned was using a 
pipette. Apart from that, we have learn how to spot the plate using the microsyringe. Besides that, we 
have also learned how to mix the mobile phase and using the cling wrap to make our beaker a complete 
chamber. In this internship, it allows us to work with various of new equipments that we never have 
an opportunity to use before. 
During the third day (9/11), we have discussed on the quantitative determination process of 
the dyes from the data that we have obtained through Thin Layer Chromatography. Not only were we 
able to explore on new equipments in the qualitative determination process , but we had also learn 
about new computer programs like ImageJ, which is typically used in gel electrophoresis, in the 
quantitative determination process. We have learned that ImageJ was able to determine the intensity of 
the dyes separated in the process of TLC. From this, it enables us to find the standard calibration curve 
which we had never done before. By using the band intensity acquired through ImageJ as our y-axis 
and using the concentration of the standard dye stock as the x-axis, a linear calibration line was 
acquired. During the day, we have also discussed about the ways to improve our TLC process and 
other possible mobile phases that would better separate the dyes and could be used during the next 
laboratory day. We have learned that in order for a research to be accurate, we have to keep on doing 
more research and repeat the experiment numerous times.  
Other days (20/11 26/12, 27/12, and 9/03) were spent on finding the errors that we have done 
and improving our experiment. At first, we have found out that the mobile phases that we initially use 
was good in separating tartrazine and ponceau 4r, but isn’t as effective in separating sunset yellow FCF 
and carmoisine. Hence, another mobile phase was needed. After conducting several experiment we 
ended up using up to fifteen different mobile phases and have chose two that we think have separate 
the four standard dyes best. The two that were chosen are: 20:12:5 butanol: distilled water: acetic acid, 
which was able to separate sunset yellow FCF and carmoisine effectively and 10:1:1 Isopropyl alcohol: 
Ammonia : Distilled Water , which was able to separate tratarzine and ponceau 4r effectively. 
During the experiment phase, it could be seen that some of the tea samples and dyes that we 
run through TLC were distorted and we had to develop ways in order to make it run nice and neat. 
Therefore, we have place toothpicks in for the plate to lean on it. We have also recognized that we 
should leave the mobile phase in the chamber for ten to fifteen minutes in advance in order to let the 
chamber be saturated with the mobile phase and wait for the samples and the standard dyes that we 
have spotted on the plate to be completely dry. Otherwise, the result won’t be able to be obtained 
successfully and accurately. 
During the quantifying determination phase, patience was highly needed as the program 
(ImageJ) requires us to start to select the spots by hand. This means that first, we need to observe the 
plate and the image of the plate obtained from scanning for any dye spots, then we need to use the 
program to select the whole spot and would have to be careful not to leave any part of the spot out as it 
would change the intensity of the spot obtained from the program and would lead to inaccurate result.  
For our learning process and working process, we think we have both success and failures. By 
doing the internship at MUIC, we had the opportunity to work on an experiment including the topic 
that we wanted to know regarding artificial food coloring in Thai tea leaves, which is relatable to our 
daily lives as Thai tea is very popular amongst people in our age. From the ability to work with the 
topic we are interested in, it allows us to work with more passion and to be more focused on the work. 
From the passion in working and the focus we had, we had learned successfully. We had learned new 
lab skills such as using the microsyringe, the pipette, the micropipette, and how to properly use, store, 
and label the chemicals that would be used in the experiment. Moreover, the fact that they are no 
preexisting paper that included the information of how to prepare tea samples properly and which 
mobile phase should be used in the process of Thin Layer Chromatography, critical thinking skill and 
the improvising skill is needed. Hence, I had learned to think critically and to improvise and come up 
with the new ideas by using the pre-existing ideas. Since this project is done in pair, communication 
skills were used. Communication is really important, and from doing this project, I had improved my 
communication skills in order to express my ideas to both the advisor and my college better. Moreover, 
during the researching phase, I had learned a lot of new information that is relevant to both the topic 
of learning and the topic that I am basing my experiment on.   
Despite the success mentioned previously, there were some mistakes and failures in 
learning. First, we had to correct the plan several times due to bad planning and due to that, time was 
lost. At first, we didn’t have an exact plan on what would we do in each day, and what would be 
needed for the day. In order to improve ourselves, we would have to think ahead in order to save time. 
Second, there were some misconceptions and misunderstandings of the task that we are supposed to 
do while we were working on the literature review, which caused us to spend more time than needed. 
In order to avoid the failure next time, discussions and questioning sessions with the advisor are 
needed in order to clear any misconceptions, to lessen the time used for completing the project and to 
be able to have deeper understanding on the topic due to the understanding of the focus.  
Despite all the success of the experiment, there could be some errors in this experiment. 
Since this experiment uses Thin Layer Chromatography and the program ImageJ, which was meant to 
be used in gel electrophoresis, the information could contain some error and might not be as accurate 
as if the were obtained by other techniques such as HPLC. Moreover, the experiment was conducted 
in only a short period of time. In order to obtain the result which is more accurate, the experiment 
should be replicated several time and the result obtained from each of the trials should be compared 
and analyzed altogether.  
As learners, we think that our strongest point is that we strive to learn and we am able to 
apply our pre-existing knowledge in order to work successfully. As we were working on the project, we 
keep coming up with different kinds of questions. When we got our questions, we noted them down 
into our notes, then we make additional researches in order to obtain my answers or ask our advisor. 
Then, we try to apply our knowledge acquired from the researches in order to improve our work. 
Moreover, we are open for any new ideas and criticism or comments about our work. We think that it 
helps us learn when the others comment on our work so that we could see our flaws and could correct 
them. However, I still have some weaknesses as a learner. We don't plan our time well when we were 
working. As we observed each other throughout the project working time, we tend to work at 
different time, which resulted in confusion of the other party sometimes as the notes and the poster, 
which is one of our final product changes from time to time without us doing anything. The flaws as 
learners are the things that we need to improve on later in our life in order to be a better learner. The 
ways to improve ourselves includes the increase in communication between each of the members of 
the team, better understanding of the topic that we would be working on and its focus in order to help 
us have a better planning in working. From doing this project, we had observed and analyzed ourselves 
and each other. Hence, we had learned about our weaknesses and strengths as learners and how could 
we improve ourselves to be better learners. 
Relating to the ESLO, this project allows us to be strategic learners since it includes learning 
and recognizing what is important to know and understand. It could be illustrated by the fact that 
most of the decisions within the lab were done by us, along with the supervision and advising of the 
advisor, Dr.Manchuta Dangkulwanich. For the decisions to be made, we need to know and recognize 
the main parts and ideas of the lab first in order to make the best decision for the experiment. It also 
relates to being innovative thinkers since it includes us building on ideas we learned from the 
preexisting papers and using the ideas to work in the lab. Since it requires reading and writing lab 
notes, we are articulate communicators. In addition, since working on this project also includes raising 
awareness of people that food dyes exists in Thai tea and there are some possible harms resulting from 
overconsumption of the dyes, it allows us to be altruistic global citizens. Lastly, since this project was 
done in pair, teamwork and common goal was made. We also worked through our differences and 
agree on a common understanding and idea. Other than that, we reflect our works and critique each 
other’s works with honesty in order to improve ourselves. Hence, both of us followed the ESLO 
‘leaders for the future’.