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

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

components, if the solvent evaporates the Rf


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