Experiment #6: Isolation of Caffeine from Tea Pre-Lab: Prepare your notebook. Include: name, partner s name, title of experiment, date, purpose, structure of caffeine, procedures (bulleted list), separation scheme (flow chart) and data (make spaces to record masses and volumes). Leave spaces for observations. 1. Why are the tea bags boiled twice? 2. Why is methylene chloride used for the extraction rather than methanol? Background: Tea has been a popular beverage for many centuries. The tea plant is native to Indochina and northern India, and wasn t widely cultivated for popular (nonmedical) use in China until around 700 A.D. Although the first confirmed medical reference is from 350 A.D., some experts say there were reports of tea being used medicinally as early as 2737 B.C. The active ingredient in tea is caffeine, one of a class of compounds called alkaloids. More specifically, caffeine is in the family called xanthines, which are among the oldest known stimulants. Caffeine stimulates the central nervous system, skeletal muscles, respiration and the heart. It is also a diuretic (it promotes urination). Caffeine is highly addictive, and is possibly the most widely abused drug in this country. Symptoms of caffeine withdrawal may include headache, lethargy, insomnia or nausea. Caffeine is also found in other plants, such as coffee, cola and cocoa (see table on next page). Tea leafs contain from 2 to 5% caffeine, along with cellulose, tannins, flavonoid pigments and chlorophylls (see structures of caffeine and tannins on next page). The caffeine can be extracted from tea using hot water. owever, the tannins, pigments and chlorophylls will also be extracted into the water. The caffeine can then be extracted from the water using methylene chloride (dichloromethane). Since the tannins are acidic, base is added to the aqueous extract in order to ionize the tannins and insure that they remain in the aqueous layer. Along with the caffeine, the chlorophylls (which are present only in small quantities) will also go into the organic layer. Thus, the caffeine that you obtain after evaporating the methylene chloride will not be totally pure. Either sublimation or crystallization can be used to further purify the caffeine. owever, we will not do that in this experiment (the caffeine is already close to pure). Decaffeinated tea (and coffee) has become more popular along with an increased public awareness of the potential health risks associated with caffeine. Traditionally, coffee and tea have been decaffeinated using methylene chloride. More recently, caffeine is often removed using supercritical carbon dioxide. owever, the term decaffeinated can be misleading, since decaffeinated tea (or coffee) can still contain up to 1.2% caffeine. In this experiment you will extract caffeine from two teas, one regular and one decaffeinated. alf of the pairs will do black tea and half will do green tea. There will be a variety of teas available in the lab, however, you may also bring your own tea to
analyze. You will calculate the % by mass of caffeine in each tea and we will compare the class results. You will also use TLC to look for caffeine in the organic layer when you extract your decaffeinated tea. TLC can show trace amounts that may not be detected by weighing. Because caffeine is a white solid, you will not be able to see it on the TLC plate by eye. owever, since caffeine is a UV-active substance (absorbs light in the ultraviolet wavelengths), we can see it by using a UV lamp. 3 C C 3 R R R R C 3 R Caffeine Glucose (R = ) Tannin (some R's = digalloyl) Catechin (a Tannin) Digalloyl group Caffeine Content of Some Foods and Drugs Coffee Decaffeinated Coffee Tea Cocoa Milk Chocolate Baking Chocolate Coca-Cola 80-125 mg per cup 2-4 mg per cup 30-75 mg per cup 5-40 mg per cup 6 mg per oz 35 mg per oz 46 mg per 12 oz can
Anacin, Midol Excedrin, extra strength Dexatrim, Vivarin Dristan o-doz 32 mg per tablet 65 mg per tablet 200 mg per tablet 16 mg per tablet 100 mg per tablet Materials: Two tea bags (one decaffeinated and one regular), methylene chloride, sodium carbonate (a 2 C 3 ), acl, 1% glacial acetic acid in acetone, distilled water, 4 beakers, watchglass cover, hotplate, 125 ml separatory funnel, test tube, TLC plate, capillary tubes, TLC chamber and UV lamp. Safety Precautions: Dichloromethane is not flammable, nor is it considered carcinogenic, but it can cause liver damage if ingested, or with repeated inhalation or skin exposures. It should be used in the fume hoods (inhalation of vapors can cause nausea or drowsiness). Avoid spills, it can also pass through both latex and nitrile gloves. Do not look directly at the UV lamp, the UV light can damage your eyes. Procedures: 1. Carefully remove the tea from the first teabag (regular), weigh it and record the mass of the tea. Put the tea back into the back and re-staple it. 2. Place the teabag and about 40 ml of distilled water into a 250 ml beaker. Cover it with a watchglass and heat to a gentle boil. Boil for 5 minutes. 3. Decant the liquid into a 150 ml beaker. Add about 20 ml more water to the teabag and boil again for 5 minutes. 4. Decant the liquid into the the 150 ml beaker (with the first liquid). Use a glass rod to gently squeeze out as much liquid as possible (without breaking the bag). Add about 1 g of a 2 C 3, swirl to dissolve, and allow the liquid to cool to room temperature. 5. Transfer the liquid to a 125 ml separatory funnel (supported with a ring clamp). Add 10 ml of methylene chloride, shake gently (vent frequently) and allow mixture to separate. Collect the organic layer in a 100 ml beaker (label and weigh the beaker first and record the mass). Add another 10 ml of methylene chloride to the separatory funnel, shake, separate and collect in the 100 ml beaker. *If an emulsion forms, try adding a little acl (1 or 2 scoops) and shake gently.
6. Discard the aqueous layer and rinse out the separatory funnel. Allow the methylene chloride to evaporate while you extract your second teabag. 7. Repeat the above procedures with the second teabag (decaffeinated), collecting the methylene chloride (step 5) in a 50 ml beaker. 8. If the methylene chloride from the first extraction (regular tea) is all evaporated, then weigh the beaker + caffeine and record the mass. If it s not dry, run a stream of air over it to complete drying before weighing (BE VERY CAREFUL TE AIR PRESSURE DES T CAUSE SPLASIG). 9. After you record the mass, scrape out a small amount of caffeine and place it in a small test tube. Add about 0.5 ml of methylene chloride. 10. Prepare a TLC plate by drawing a line about 1 cm from the bottom of the plate, and then marking 3 places on the line for spotting. btain a TLC chamber and two capillary tubes. Place about 8 ml of TLC solvent (1% glacial acetic acid in acetone) into the chamber. 11. Spot the plate with your samples. First, spot the left and center marks with your first tea extract (one spot each). ext, with a new capillary tube, spot the second tea extract (from the 50 ml beaker) on the right-hand mark. Spot 3 or 4 times, allowing the spot to dry between each (this solution is more dilute). Then spot the second tea extract in the same way over the center mark, right on top of the first spot. Label the spots under the marks. 12. Develop the TLC plate and mark the solvent line before drying it. bserve the plate under short-wave UV light and circle any spots you see. ote the appearance of the spots (bright, dark, colored, etc.). Make a sketch of the plate (with observations) in your notebook. Measure the Rf of each spot. 13. If the methylene chloride from the second extraction (decaffeinated tea) is all evaporated, then weigh the beaker + caffeine and record the mass. If it s not dry, run a stream of air over it to complete drying before weighing. 14. Calculate the mass of the caffeine obtained from each tea, and then calculate the % by mass of caffeine for each tea. Waste Disposal: Teabags can go into the trash. Aqueous solutions should go into the aqueous waste container (they contain base). Any organic chemicals should go into the organic waste container. Capillary tubes go into the glass waste (not the trash).
Report: 1. In your notebook, make tables for your results. ne should include the brand of tea, regular or decaffeinated, mass of tea, mass of caffeine and % by mass of caffeine in the tea for each tea tested (two in this case). The other should include the R f values and appearance for each spot for each of the three lanes on your TLC. 2. Write a brief discussion of the experiment. In this section you should summarize your results (brand, regular or decaffeinated and % by mass caffeine) and note any interesting observations (odor, color etc.). Also summarize your TLC results and discuss their relevance. Make any possible conclusions about the experiment (successful vs. unsuccessful and reasons why; compare your results to expected results). 3. Answer the post-lab question. 4. Make a copy of your lab notebook pages (do not tear out the originals) and attach the question sheet with your answers. 5. Staple the discussion, post-lab question notebook page copies and upcoming pre-lab together. Post-Lab Questions: 1. Before extracting the caffeine with methylene chloride, you added a 2 C 3 to the aqueous portion. Explain why. Also, write the chemical reaction that would occur between phenol and a and incorporate it into your explanation. 2. Caffeine is soluble in methanol, as well as methylene chloride. Why couldn t you use methanol to extract the caffeine from the aqueous portion?