Tlc.pdf

Chem 35.1 – TEG

Espiritu, Walter Aljhon Silong, Rafaelle Tumimbang, Glenn Vincent

March 4, 2014

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 12mm 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

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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. 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 Erlenmeyer flask, then put the crude in a roundbottom bottle and add 10mL dichloromethane and reflux the solution. Boil the solution for 4 minutes and pour the supernatant to the filtrate. Repeat this step thrice. Then collect all filtrate in separatory funnel and add 10mL saturated NaCl 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.)

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.

Figure 1. Structure of acetaminophen

Acetaminophen (paracetamol) is a pain-relieving analgesic and antipyretic (fever-reducing) agent. The structure is highly polar due to a phenoxy moiety that is why it can be deduced that it has no affinity towards the non-polar solvent.

Figure 2. Structure of caffeine

V. Results and Discussion The following are the results of the chromatogram for analgesics, the retention factor (Rf) value is computed as follows: 𝑅𝑓 =

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒 (𝑐𝑚) 𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑐𝑚)

Compound

Distance travelled (cm)

Rf value

Acetaminophen

0

0

Caffeine

5

0.862

Unknown

6

1.03

Solvent

5.8

xxx

Table 1. Experimental Rf values of different analgesics.

However, caffeine, found on coffee and known for its physiological effect on the body, is highly nonpolar. Its high Rf value is due to its high affinity to the nonpolar solvent. As a result, it can be inferred that the unknown solution is also nonpolar due to high Rf value. On the other hand, these are the results for the isolation of lycopene. Spot

Distance travelled (cm)

Rf value

Filtrate Residue

0 5.9

0 1.035

Filtrate + Residue

5.9

1.035

Solvent

5.7

Table 2. Rf values for isolation of lycopene

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

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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 Page 3

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

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