Caffeine Extraction from Thea sinensis and Determination of purity by Thin Layer

Chromatography

Charmaine Almonte, Arnold Balais Jr., Jan Michael Eli Bautista*, and Arvin Raphael
Bayot

Department of Biological Sciences, University of Santo Tomas, Manila, Philippines

ABSTRACT
Tea leaves (Thea sinensis) contain about 15-35mg of alkaloid caffeine which is known to have a
mild stimulating effect to the central nervous system. In the experiment, the group extracted, isolated and
characterized the caffeine present in the leaves. The members applied numerous steps in order to obtain
the results. The calculations are done through following a formula which then led to a 0.72% percent yield
of crude caffeine. In addition, the group performed Thin Layer Chromatography in order confirm the purity
of the crude caffeine.

INTRODUCTION

Molecular Structure for Caffeine

Coffee is a staple of any college student, as it provides a way to be able to take

away the drowsiness that accompanies late night readings, projects, and the like. A
study by Nicole Olsen (2013)[1] indicates that in the University of New Hampshire
Students, sixty-six percent of respondents from the survey regarding caffeine
consumption were from 18-21 years old. This demographic emphasizes the tendencies
of college students to consume caffeinated products in this foreign university. Despite
being abroad, those in the same age group here in the Philippines can be said to follow
the same trend based on personal experiences from the group. The substance caffeine
is responsible for the stimulating effects of coffee, as it blocks the adenosine receptors
in the brain, making the body unable to relax which is the purpose of this hormone [2]. It
is no surprise then that Coffee is a go-to drink of people who have a huge work load to
be done in the evening because it allows them to keep awake. It should be noted that
continuous consumption of caffeine disturbs the bodys normal sleep cycle and that
after some time, the person will become resistant to its effects. A negative effect,
however, is that the consumer will also become reliant on coffee, developing an
addiction, and suddenly stopping in drinking it, will result in a withdrawal syndrome.

However, caffeine is not only commercialized within products such as coffee, but
also in tea leaves. Teas contain about 15-35 mg worth of caffeine, in contrast to the
average of 135 mg worth in coffee [3]. In this experiment, our group aims to isolate,
purify, and characterize the caffeine extracted from Lipton tea leaves, calculate the
percentage yield of this product, and finally testing its purity via Thin Layer
Chromatography.

METHODOLOGY
Three bags of tea were distributed to each group. The bags were emptied and
placed in a tared 50 mL beaker then the weight of the tea leaves was obtained. The tea
leaves were placed back in the tea bags. The tea bags were put in a boiling 100 mL
distilled water and let it boiled for 5 minutes. Next, the hot tea extract was cooled to
room temperature and was placed in a separatory funnel with 20 mL DCM
(dicholoromethane) to produce DCM extract (a) and Tea extract.

The tea extract was extracted with fresh 20 mL DCM which will then produce
DCM extract (b) and tea extract. The DCM extract (b) was combined with DCM extract
(a) to give 40 mL DCM extract. While the tea extract was extracted with another fresh
20 mL DCM which will produce DCM extract (c) and another tea extract that will be
discarded. The 40 mL DCM extract was combined with DCM extract (c) to give 60 mL
DCM extract. The 60 mL DCM extract was transferred using a rinsed separatory funnel
with 20 mL 6M NaOH extract to make an aqueous and DCM layer. The DCM layer was
dried with anhydrous Na2SO4 (sodium sulfate) and decanted to an evaporating dish
until the next meeting. Next, the dried caffeine was collected in a vial by using a micro
spatula. The percent yield of the caffeine was computed and then, a pea sized portion
of the caffeine was placed inside a calibrated 2 mL test tube, which contained 1 mL of
distilled water. It was later blotted on a particular point (point B) on a TLC plate, and
point A was blotted with pure caffeine. This was done in order to test its purity by
comparing the number of bubbles formed on the side of the pure caffeine against the
crude caffeine obtained by the group.

RESULTS

Part A

Data Sought Value

Weight of Tea Leaves 6.2311gg
Weight of empty evaporating dish 104.21377g
Weight of evaporating dish and crude caffeine 104.2587g
Weight of crude caffeine 0.045g
Percent yield of crude caffeine 0.7222%
Density of DCM 1.33 g/L
Density of Water 1.0 g/L

Appearance (Extract) = black and powdery

Appearance (Crude Caffeine) = translucent and thin film

Part B

TLC plate

Spots for standard caffeine = 1

Spots for crude caffeine = 1

DISCUSSION

In order to discuss the results presented in the previous pages, it is important to

understand some terms and concepts. The basic property of caffeine comes from the
lone pair of electrons found around the nitrogen(s). It is an achiral molecule and does
not have any stereoisomers. Caffeine is also a polar molecule; this is evident because
of the london dispersion forces, dipole-dipole interactions, and hydrogen bonding
present when it is in water. It also has a very hydrophobic region. The nitrogen present
in caffeine controls solubility. Caffeine is soluble in water at approximately 2.2 mg/ml at
25C, 180 mg/ml at 80 C, and 670 mg/ml at 100C. It is an organic molecule that has
the properties of an organic amine base. When extracting caffeine from the tea leaves,
the water was kept at a high temperature in order to increase solubility of caffeine in
water to about 670 mg/ml at 100C.
 Extraction Set - Up

Another concept of the experiment should be discussed is the use of DCM of

dichloromethane in order to extract the caffeine. The solution of these dissolved
compounds is referred to as the Extract. Here the organic
solvent dichloromethane is used to extract caffeine from an aqueous extract of tea
leaves because caffeine is more soluble in dichloromethane (140 mg/ml) than it is in
water (22 mg/ml). But before the addition of dichloromethane, the tea solution was first
cooled down to room temperature (~25 C) because dichloromethane has a boiling
point of 40C. If the solutions temperature was not lowered, the dichloromethane would
evaporate and the caffeine would not be properly extracted

The last concept of the experiment is the Thin Layer Chromatography. Thin-layer
chromatography involves the same principles as column chromatography, it also is a
form of solid-liquid adsorption chromatography. In this case, however, the solid
adsorbent is spread as a thin on a plate of glass or rigid plastic. A drop of the solution to
be separated is placed near one edge of the plate, and the plate is placed in a
container, called a developing chamber, with enough of the eluting solvent to come to a
level just below the "spot." The solvent migrates up the plate, carrying with it the
components of the mixture at different rates. The result may then be a series of spots
on the plate, which means that the caffeine was not pure. However, in the results, it is
seen that our crude caffeine is in fact, pure caffeine since its TLC result was equivalent
with one another.

CONCLUSION
Caffeine is indeed found in tea leaves. It is confirmed by making use of
Dichloromethane to extract the product of interest from the substrate, and it is left to
sublime on an evaporating dish. The caffeine extracts purity is also confirmed by
comparing the bubbles formed on a TLC plate, where in points A and B contained a
drop of pure and crude caffeine respectively.

REFERENCES
[1] Retrieved February 18, 2017 from the World Wide Web:
http://scholars.unh.edu/cgi/viewcontent.cgi?article=1102&context=honors

[2] Retrieved February 18, 2017 from the World Wide Web: http://health.howstuffworks.com/wellness/drugs-alcohol/caffeine-
awake.htm

[3] Laboratory Experiments in Organic Chemistry. Carlos P. Garcia. University Publishing House, Manila, 2005.