Caffeine isolation from tea leaves

By
Krittin Tansida 5961119
Sakornchan Mattariganont 5961143
Viriya Unchaleevilawan 5961085

Advance Chemistry 12
Ms. Sorasaree Tonsiengsom

Mahidol University International Demonstration School

Semester 1 Academic Year 2018-2019
Abstract:
The lab that we are doing is extracting caffeine out of the tea solution by doing caffeine
isolation. We then use thin-layer chromatography technique(TLC) to give support that there's
really caffeine in our sample that we extract out from the tea solution by referring with the
retention factor caffeine references with our sample. Finally, we use the sample to also check the
caffeine melting point to determine the purity of our caffeine sample which the result was that
our sample is not pure caffeine but there is caffeine in the sample we extracted.

Introduction:
Caffeine is a common substance that can be found in coffee, tea, and Kola nuts. And the
tea leaves contain more caffeine than coffee beans, which tea leaves contain about three percent
of content by weight (Teatulia, 2018). The caffeine substance has a huge effect to our body if
consumed, it acts as a central nervous system stimulant, which makes people feel more awake
and less tried. So this substance is a common ingredient in medication, for example, treatment of
a headache, migraine and many others (Pietrangelo A. and Cherney K, 2007). On the other hand,
caffeine also has a positive effect. If we consumed caffeine about 200 mg, it can boost our
long-time memory. Moreover, it also reduces the risk of liver, mouth and sore throat cancer. And
protect type 2 diabetes, Parkinson's disease, cardiovascular disease and stroke (Honor W, 2015 ).

Figure 1(from the left) illustrates the top layer which is aqueous solution and the bottom layer which the
solution of caffeine and dichloromethane.
Figure 2 illustrates the water (the bot and the solution of caffeine and dichloromethane.
 Extraction is a technique that used to separate an organic compound from the mixture.
Which in this lab, we used the method of extraction to extract tea solution by using
dichloromethane and separatory funnels to separate the layers of caffeine with dichloromethane
and tea solution. We used dichloromethane to extract caffeine instead of water because caffeine
is more soluble than water (Amrita, 2018). And after we finished all process, we have measured
the purity by using the TLC process, which is the thin layer chromatography. TLC is a process
that used for separate the mixture, which contains the stationary phase and mobile phrase (Clark
J, 2016). The stationary phase that we used to extract the caffeine is Silica gel, and the mobile
phrase that travels up through the silica gel contains ethyl acetate, hexane, and acetic acid. And
we can get the results by looking at the mobile phrase run through the stationary phrase that
carries the component of a mixture with it, and we can see the different component travel at the
different phrase. Moreover, TLC also tells us that which component is polar and non-polar,
which higher retention factor is non-polar and the lower retention factor is polar (Clark J, 2016).
another method of measurement that we used in the lab is comparing the melting point between
the reference and our sample.

Objective:
The purpose is this lab is to isolate the caffeine from the tea leaves. When we finish
isolating the caffeine then we will identify melting point and confirm purity of caffeine. Lastly,
we will find the Retention factor to check how identical is our tea leave caffeine with the
reference caffeine.

Materials:
1. TLC silica gel
2. UV lamp
3. Melting point determination machine
4. Thermometer
5. Separatory funnel
6. Capillary tube
7. 45 ml Dichloromethane solution
8. 20 ml tea solution
9. 40 ml of water
10. Sodium sulfate (Na2 SO4)
11. Rotoevaporation machine

Procedure:

Caffeine Extraction
1. Prepare concentrated tea solution
 2. Mix tea solution with 15 mL dichloromethane in the separatory funnel
3. Place the separatory funnel on the hand and shake the bottle in C-direction for 3-4 times.
4. There are two separated layers which are aqueous and organic layer. Open tap to pour the
organic layer into the erlenmeyer flask and collect aqueous solution into separatory
funnel.
5. Do step two to four again for 3 times to make sure that every caffeine is extracted.
6. After finish the fifth step, pour out the remaining aqueous layer to sink.
7. Wash the remaining solution with 20 mL of 6 M NaOH in separatory funnel.
8. Wash the remaining solution with 20 ml of 6 M H2O in separatory funnel.
9. Collect organic layer to erlenmeyer flask and discard aqueous layer.
10. Dry over Na2SO4
11. Decant into 100 mL round bottom
12. Rotovap to dryness
13. Collect Pure caffeine into the plate

Testing caffeine by TLC chromatography

1. Prepare silica chromatography paper
2. Measure 1.5 cm from the width side and mark the line
3. Measure 1 cm from the opposite side and mark the line again.
4. Write two separate dots on the first line. Classify two dots by write down which dots are
reference (caffeine from the lab) and sample.
5. Drop the solution of caffeine from the lab on reference dot and the solution from the real
sample on sample dot.
6. Put paper on the solution of dichloromethane and leave it until the solution runs through
the line.
7. After the solution reaches the line, use UV lamp to observe how far solution reaches
8. Collect data by drawing circle around the longest spot on silica paper
9. Measure Rf of each two substances in paper

Testing for melting point of caffeine

1. Put reference and sample into different capillary tubes
2. Warm rotoevaporatory machine until 200 degree celsius.
3. Put capillary tubes that contain reference and sample to the rotoevaporatory machine
4. Observe them until it melts
5. Collect temperature data from when it melts until
Results :

Figure 3 (from the left) illustrates Rf value of each group (our group is in the middle)
Figure 4 illustrates the process of melting reference and sample before reaches 200 degree Celsius
Figure 5 illustrates how Rf value measure

According to our experiment, we were able to extract a caffeine out of our tea solution
for later use of finding it melting point, Retention factor, and how identical our caffeine sample
with a pure caffeine by using TLC silica gel.
For our results, The Thin Layer Chromatography (TLC) illustrates that we have caffeine
in our mixture by comparison with the caffeine references, and it also shows that our sample is
mixture, because our sample contain more than one dot. For the melting point, our references got
229 degree celsius and our sample got 210 degree celsius. And we can see from the results that
our sample and references has a different melting point, which means our caffeine is not purity.
The measurement of the caffeine sample TLC silica gel is 1.5 centimeters from the
baseline(distance sample) and the length from the baseline to the finish line(distance solvent) is 5
centimeters. Finally, after doing the calculation to find the retention factor of the sample and
reference we get the same result which is 0.3.

Discussion:
The emulsion will be found as the middle layer during liquid-liquid extraction. To avoid
the mixing of emulsion, the separatory cannot be shake vigorously, so two separated layer cannot
be mixed. However, the simple method to destroy make it completely separated is to slowly
shake separatory funnel into C-shaped direction and open valve to release the pressure of
dichloromethane inside the funnel. Drying agent or Na2SO4 is very important for this
experiment. Caffeine is not the only substance in tea solution, but also tannins which are soluble
in dichloromethane same as caffeine are in the solution. In order to separate tannins from
caffeine, Sodium sulfate is involved to transform tannins to phenolic anions which is insoluble in
dichloromethane but more soluble in water. When the water washes mix with the solution,
phenolic anions or tannins will dissolve into water and pour off with water.
To prove that caffeine from the experiment is pure, Thin Layer Chromatography (TLC)
and rotoevaporatory machine are required in this experiment. TLC examine the solubility of
caffeine in dichloromethane, and rotoevaporatory machine tests the melting point. From the
result, the solubility of reference and sample are the same that the Rf is 0.3. But moving to the
result from rotoevaporatory machine, the melting point is slightly different that the melting point
of reference and sample are 229 and 210 degrees Celsius, respectively. However, Measuring
melting point is not precise. In the experiment, the melting point of both sample and reference
are measured as one value, but in fact, the data has to be collected as a range by measuring from
the substance starts to melt until it becomes all liquid. As the result, we cannot say the exact
conclusion that it is pure or not, but the product is mostly pure comparing two Rf value together.
To find the intermolecular force of molecules, the polarity of substances need to be
considered. According to Edwards Q., Lunat I., Garner-O’neale L., Kuliko S. (2015), caffeine is
a polar molecule which is soluble in water because the greater electronegativity of oxygen on
carbonyl group bend most of electrons toward carbonyl group. So, the caffeine which is polar
molecule has both dispersion force and dipole-dipole force. However, these two forces cannot be
strong enough to pull each molecules together in above 200 degree Celsius. The hydrogen bond
is the strongest bond in the high melting point. Due to the structure of caffeine, there are
hydrogen bond in N-H and O-H bond.

Calculation:

distance sample
Retention factor = distance solvent

Distance sample = length of base line to the location of the dot= 1.5 cm
Distance solvent= length of base line to the finish line =5 cm

Retention factor(reference) = 1.5 cm

5 cm = 0.3
Retention factor(sample) = 1.5 cm
5 cm = 0.3

The retention factor of reference and the sample are the same (Rf​(ref)​=Rf​(sample)​)
 Conclusion:
In conclusion, We used extraction technique to extract the caffeine out of tea solution, by
using dichloromethane and separatory funnels. And after we got our sample, we have measured
our sample purity and checked that our sample contain caffeine, by using chromatography and
measured melting point method. Which our results show that our sample is a mixture, which
contain caffeine. And our sample melting point is different from the references, accounting for
210 and 229 degree celsius.

References:

Pietrangelo A. and Cherney K (2007), Effects of caffeine, retrieved from;

https://www.healthline.com/health/caffeine-effects-on-body#1

Teatulia (2018), Caffeine in Tea, retrieved from;

https://www.teatulia.com/tea-101/caffeine-in-tea.html

Amrita (2018), Extraction caffeine from Tea, retrieved from;

http://vlab.amrita.edu/?sub=3&brch=64&sim=169&cnt=1

Honor W. (2015), How Caffeine affects your health, retrieved from;

https://www.medicalnewstoday.com/articles/271707.php

Clark J. (2016), Thin layer chromatography, retrieved from;

https://www.chemguide.co.uk/analysis/chromatography/thinlayer.html

(Clark J, 2016), Paper chromatography, retrieved from;

https://www.chemguide.co.uk/analysis/chromatography/paper.html

Edwards Q., Lunat I., Garner-O’neale L., Kuliko S. (2015), Distribution of Caffeine Between
Selected Water-Organic Solvent Media, Int. J. Chem. Sci.: 13(3), 2015, 1218-1226, Retrieved
from
https://www.tsijournals.com/articles/distribution-of-caffeine-between-selected-waterorganic-solv
ent-media.pdf