Chem 35.

1 – TEG

Espiritu, Walter Aljhon March 4, 2014

Silong, Rafaelle
Tumimbang, Glenn Vincent

I. Abstract
Chemicals that are responsible for natural effects such as the green color of the leaves and brown
color of human skin are often studied through isolation and separation of these compounds from natural
sources. Chemists, particularly organic chemists, devised many different methods of separation, isolation,
and purification of organic compounds. Examples of these methods are fractional distillation of liquids and
recrystallization of solids. Some other methods are more accurate and appropriate when separating small.
In this experiment, one of the most useful, accurate and appropriate but cheap method of separation and
isolation is explored which is chromatography.

II. Keywords: lycopene, thin-layer chromatography, mobile phase, stationary phase, polarity
III. Introduction
Naturally-occurring compounds in living
organisms, often giving distinct characteristics
such as the color of the leaves, fruits, and even
animals, are carefully studied through various
isolation and separation means. Chromatography
is one of the most commonly-devised methods of
separation due to its accuracy and feasibility. An
example of which is thin-layer chromatography,
used to separate non-volatile mixtures and to
qualitatively observe and monitor organic
reactions. It uses a stationary phase, a medium in
which the mobile phase travels, carrying the
components of the mixture with it. Different
compounds travel at different rates and distances.
This is mainly used to compare and identify a
separated compound from the mixture.
Lycopene is a bright red carotene pigment
that is commonly found in tomatoes and other red
fruits and vegetables. It is an important
biosynthetic intermediate, and is a valuable
organic compound due to its health benefits. To
study this compound requires isolation from its
natural source, thus using various laboratory
techniques to successfully separate it.
An analgesic is a drug used to relieve
pain. It acts in the nervous system (central and
peripheral), which reversibly eliminates sensation.
Usage of these drugs depends on the severity
and response to other medication products, in
most cases, starting from ones with mild effects.
IV. Methodology
Major Step 1
Prepare the standard solutions: aspirin,
acetaminophen, ibuprofen, caffeine and unknown
(10 mL, 1% solution in ethanol.) The unknown is
prepared by crushing a part of a tablet and adding
it to a reaction tube or small vial with enough
ethanol to make a 1% solution. Then, prepare a
developing chamber by placing a folded paper
lengthwise in a wide mouth bottle. Prepare 10mL
of 99:1 mixture of ethyl acetate and acetic acid to
use as eluant, then add an amount of eluent to
the developing chamber so that it forms a 1-cm
layer on the bottom of the container. Screw the
cap tightly and shake container well. This is done
to saturate the atmosphere of the interior of the
chamber with the solvent. Then, obtain a 6x10 cm
strip of silica gel chromatogram sheet and place a
pencil dot in the middle of the sheet about 1 cm
from one end. Using a capillary tube, apply a spot
of pigment solution over the pencil dot by lightly
and briefly applying the tip of the tube to the
surface of the plate, apply spot four or five times,
do not allow the spot to diffuse for more than 1-
2mm in diameter. When the spot has dried, place
the strip in the developing chamber (the spot must
be above the solvent level), allow the solvent front
to move within 2-3mm of the top of the strip then
remove the strip and mark the position of the
solvent with a pencil and allow the plate to dry.
Then, set the plates on a paper towel to dry once
they have been removed from the chamber.

Expt. 3: Analysis of Analgesics and Isolation of Lycopene by TLC Page 1

Chem 35.1 – TEG

Finally, prepare a small jar containing iodine
crystals and insert one plate at a time. Cap the
container and warm it gently on a steam bath until
the spots begin to appear. Notice which spots
become visible and note their color.
The solvent used, ethylacetate and acetic
acid mixture, is highly polar. Therefore, the
observed values are expected. This can also be
explained through the structures of the solutes.

Major Step 2
Transfer 5-g sample of tomato paste to
the bottom of a 50-m: beaker followed by 10mL of
acetone, add 10mL ethanol then heat for 5
minutes. Then, filter it with filter paper and press
to take of all filtrate. Keep the filtrate in a 125mL Figure 1. Structure of acetaminophen
Erlenmeyer flask, then put the crude in a round-
bottom bottle and add 10mL dichloromethane and Acetaminophen (paracetamol) is a pain-relieving
reflux the solution. Boil the solution for 4 minutes analgesic and antipyretic (fever-reducing) agent.
and pour the supernatant to the filtrate. Repeat The structure is highly polar due to a phenoxy
this step thrice. Then collect all filtrate in moiety that is why it can be deduced that it has no
separatory funnel and add 10mL saturated NaCl affinity towards the non-polar solvent.
solution. Shake gently and allow separation.
Collect the lower layer, then add 1 teaspoon of
anhydrous Na2SO4 and allow to stand for 5
minutes. Filter the solution and keep the filtrate in
a dark bottle away from t\light to prevent the
disappearance of the color of lycopene. Finally,
isolate lycopene by TLC technique (in this case
develop the plate with 80:20 hexane-acetone
mixture.)
Figure 2. Structure of caffeine
V. Results and Discussion
The following are the results of the However, caffeine, found on coffee and
chromatogram for analgesics, the retention factor known for its physiological effect on the body, is
(Rf) value is computed as follows: highly nonpolar. Its high Rf value is due to its high
affinity to the nonpolar solvent.
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑐𝑚) As a result, it can be inferred that the
𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑐𝑚) unknown solution is also nonpolar due to high Rf
value. On the other hand, these are the results for
the isolation of lycopene.
Distance
Compound Rf value Distance
travelled (cm) Spot Rf value
travelled (cm)
Acetaminophen 0 0 Filtrate 0 0
Caffeine 5 0.862 Residue 5.9 1.035
Unknown 6 1.03 Filtrate +
5.9 1.035
Solvent 5.8 xxx Residue
Table 1. Experimental Rf values of different analgesics. Solvent 5.7
Table 2. Rf values for isolation of lycopene

Expt. 3: Analysis of Analgesics and Isolation of Lycopene by TLC Page 2

Chem 35.1 – TEG
It can be observed that the compound on
the residue and the filtrate + residue is highly
nonpolar due to its high Rf value, and therefore
has high affinity towards the nonpolar solvent, in
this case, the hexane-acetone mixture, which
affirms the presence of lycopene in both samples.
VI. Guide Questions
1.Compare thin-layer chromatography with
column chromatography with regard to (i) quantity
of material that can be separated, (ii) the speed,
(iii) the solvent systems, and (iv) the ability to
separate compounds.
In chromatographic terms, TLC has a big
advantage over other chromatographic
techniques. TLC can perform multiple analyses
simultaneously.
2. What problem will ensue if the level of the
developing liquid is higher than the applied spot in
a TLC analysis?
If the developing liquid is higher than the
applied spot in TLC the spot may be washed off
and lost. It is also possible that the spot will not
move up the plate but spread out and
contaminate the solvent in the jar.
3. In what order (from top to bottom) would you
expect to find naphthalene, butyric acid, and
phenyl acetate on a silica gel TLC plate
developed with dichloromethane?
Since the stationary phase is silica, which
is polar, the least polar substance will travel the
highest. So the order, from top to bottom, is
naphthalene, phenyl acetate and butyric acid.
4. Why is it necessary to run TLC in a closed
container and to have the interior vapor saturated
with the solvent?
TLC needs to be run in a closed container
because it needs to maintain an atmosphere with
a saturated solvent. Saturating the atmosphere in
the container with vapor stops the solvent from
evaporating as it rises up the plate. It is also done
to ensure maximum resolution between
Expt. 3: Analysis of Analgesics and Isolation of Lycopene by TLC
components, if the solvent evaporates the Rf
value would be lower than expected.
5. What will be the appearance of a TLC plate if a
solvent of low polarity is used in the
development? Too high polarity?
At a low polarity the spots will stay on or
near the origin. On a too high polarity the spots
will be at the top of the plate.
6. Discuss the importance of lycopene?
Lycopene is a naturally occurring chemical
that gives our fruits and vegetables its redness. It
is a red, fat-soluble pigment found in certain
plants and microorganisms, where it serves as an
accessory light-gathering pigment and protects
them from ultraviolet B radiation and it can be
found mostly in tomatoes and tomato products.
Lycopene is believed to help prevent heart
diseases and cancer like cancer of the prostate,
colon, breast, lungs, bladder, ovaries and
pancreas. It is also believed that lycopene can
treat HPV (human papilloma virus) infections.
Lycopene has also been found effective in the
treatment of eye diseases, male infertility,
inflammation, and osteoporosis. There are still
many studies that are being conducted to prove
lycopene’s role in cancer prevention and its
benefits to the human body.
VII. Conclusion and Recommendation
The experiment showed that the unknown
solution is a non-polar compound due to its high
Rf in comparison to caffeine. The stationary
phase in the experiment is the silica gel, which is
polar. It has a low affinity to a polar medium
compared to the analgesics that were analyzed.
The presence of lycopene in the second
part of the experiment was also observed due to
its high affinity to non-polar solvent, in this case,
the hexane and acetone mixture.
VIII. References
Carey, F. (2006). Organic Chemistry, 6th
Edition
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Chem 35.1 – TEG

Klein, D. (2012). Organic Chemistry.

Thin-layer chromatography. Retrieved

from:http://www.chemguide.co.uk/analysis
/chromatography/thinlayer.html. 2007.

Willette, R. Analgesic agents. Retrieved

from:http://chemistry.ncssm.edu/mc/opiate
s/resources/will.pdf

I hereby certify that I substantially contribute to

this report.

_____________________
Walter Aljhon Espiritu

_____________________
Rafaelle Silong

_____________________
Glenn Vincent Tumimbang

Expt. 3: Analysis of Analgesics and Isolation of Lycopene by TLC Page 4