Macroscale and Microscale Organic Experiments SIXTH EDITION Macroscale and Microscale Organic Experiments, Sixth Edition

Macroscale and Microscale Organic Experiments SIXTH EDITION

Kenneth L. Williamson Mount Holyoke College, Emeritus

Katherine M. Masters Pennsylvania State University

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Macroscale and Microscale Organic Experiments, Sixth Edition Kenneth L. Williamson, Katherine M. Masters Publisher: Charles Hartford Senior Development Editor: Sandra Kiselica Editorial Assistant: Jon Olafsson Associate Media Editor: Stephanie Van Camp

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Recrystallize the products from the minimum quantities of hot hexanes. Isolate the crystals, dry them, and determine their weights and melting points. Calculate the percent recovery of the crude and recrystallized products based on the 45 mg quantity of each in the original mixture. The TLC plate is eluted with a 30:1 mixture of toluene and absolute ethanol. Do you detect any contamination of one compound by the other? Cleaning Up. If regulations allow, empty the chromatography column onto a piece of aluminum foil in the hood. After the solvent has evaporated, place the alumina and sand in the nonhazardous waste container. Otherwise, place the wet alumina and sand in a designated waste container. Evaporate the crystallization mother liquor to dryness and place the residue in the hazardous waste container.

5 . I S O L AT I O N O F LY C O P E N E AND ␤-CAROTENE

FIG. 9.10 Evaporation of a low-boiling liquid under vacuum. Heat is supplied by the hand, the contents of the flask are swirled, and the vacuum is controlled with the thumb.

Lycopene, the red pigment in tomatoes, is a C40-carotenoid made up of eight fivecarbon isoprene units. β-Carotene, the yellow pigment of the carrot, is an isomer of lycopene in which the double bonds at C1—C2 and C'1—C'2 are replaced by bonds extending from C1 to C6 and from C'1 to C'6 to form rings. The chromophore in each case is a system of 11 all-trans conjugated double bonds; the closing of the two rings renders β-carotene less highly pigmented than lycopene. These colored hydrocarbons have been encountered in the TLC experiment (see Chapter 8). The isolation procedure described here affords sufficient carotene and lycopene to carry out analytical spectroscopy and some isomerization reactions. It might be of interest if some students isolate carotene from strained carrot baby food while others isolate lycopene from tomato paste. Lycopene is responsible not only for the red color of tomatoes but also of red grapefruit, watermelon,, and flamingos. If flamingos do not include foods containing lycopene in their diet, they will be white. Lycopene is the predominant carotenoid in blood plasma. It is not converted into vitamin A as carotene is, but it is a powerful antioxidant and is an efficient scavenger of singlet oxygen.

Isoprene

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Macroscale and Microscale Organic Experiments

Carotenoids are highly sensitive to photochemical air oxidation; therefore, protect solutions and solids from undue exposure to light and heat and work as rapidly as possible. Do not heat solutions when evaporating solvents and, if possible, flush apparatus with nitrogen to exclude oxygen. Research workers isolate these compounds in dimly lit rooms and/or wrap all containers and chromatographic columns in aluminum foil, and carry out extractions and crystallizations using solvents that have been deoxygenated.

Dehydration and Extraction of Tomato or Carrot Paste IN THIS EXPERIMENT, a vegetable paste is stirred with acetone to remove water, but not the coloring matter, from the paste. The mixture is filtered, the yellow filtrate discarded, and the material on the filter squeezed as dry as possible. This solid is then extracted three times with dichloromethane. The solution is dried over calcium chloride and evaporated at room temperature under vacuum to leave the crude carotenoids.

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Video: Column Chromatography; Photo: Column Chromatography

Add 5 g of tomato or carrot paste to a 15-mL centrifuge tube or 25 ⫻ 150-mm test tube; then add about 7 mL of acetone and stir the paste for several minutes until it is no longer gummy. This acetone treatment removes most of the water from the cellular mixture. Filter the mixture on a small Büchner funnel. Scrape out the tube with a spatula, let it drain thoroughly, and squeeze out as much liquid as possible from the solid residue in the funnel with a spatula. Discard the yellow filtrate. Then return the solid residue to the centrifuge tube and add 5 mL of dichloromethane to effect extraction. Cap the tube and shake the mixture vigorously. Filter the mixture on a Büchner funnel once more, repeat the extraction and filtration with two or three further 5-mL portions of dichloromethane, clean the tube thoroughly, and place the filtrates in it. Dry the solution over anhydrous calcium chloride pellets, filter the solution into a small flask, and evaporate the solution to dryness with a stream of nitrogen or under vacuum using a rotary evaporator (Fig. 9.8 on page 197) or the apparatus shown in Figure 9.10 on page 201, never heating the sample above 50°C. Determine the weight of the crude material. It will be very small. If the residue is dry, as it should be, add just enough dichloromethane to dissolve the residue. Save 1 drop of this solution to carry out a TLC analysis (using dichloromethane as the eluent on silica gel plates; see Chapter 8). Then add 200 mg of alumina to the remaining dichloromethane solution and evaporate the mixture to dryness, again without heat.

Column Chromatography The crude carotenoid is to be chromatographed on an 8-cm column of basic or neutral alumina, prepared with hexanes as the solvent (see the detailed procedure at the beginning of this chapter). Run out excess solvent or remove it from the top of the chromatography column with a Pasteur pipette. Using the dry sample loading method described at the beginning of this chapter, add the 300 mg of alumina that has the crude carotenoids absorbed on it. Add a few drops of hexanes to wash down the inside of the chromatography column and to consolidate the carotenoid mixture at the top of the column. Elute the column with hexanes, discard the initial colorless eluate, and collect all yellow or orange eluates together. Place a drop of



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solution on a microscope slide and evaporate the remainder to dryness using a stream of nitrogen or a rotary evaporator (see Fig. 9.8 on page 197). Examination of the material spotted on the slide may reveal crystallinity. If you are using tomato paste, a small amount of yellow Gr beta-carotene will come off first, followed by lycopene. Collect the red lycopene separately by eluting with a mixture of 10% acetone in hexane and also evaporate that solution to dryness. Finally, dissolve the samples obtained by evaporating the solvent in the least possible amount of dichloromethane and carry out TLC of the two products in order to ascertain their purity (see Chapter 8, Experiment 2). You may want to combine your purified products with those of several other students, evaporate the solution to dryness, dissolve the residue in deuterochloroform, CDCl3, and determine the 1H NMR spectrum (see Chapter 12). Also obtain an infrared spectrum and a visible spectrum (in hexane). Note that Gr beta-carotene is in demand as a source of vitamin A and is manufactured by an efficient synthesis. Until very recently no use for lycopene had been found. Cleaning Up. Place recovered and unused dichloromethane in the halogenated organic waste container; the solvents used for TLC in the organic solvents waste container. If local regulations allow, evaporate any residual solvent from the drying agents in the hood and place the dried solid in the nonhazardous waste container. Otherwise, place the wet drying agent in a waste container designated for this purpose. Used plant material and dry TLC plates can be discarded in the nonhazardous waste container.

For Further Investigation The carotenoids of any leaf can be isolated in the manner described in this experiment. Grind the leaf material (about 10 g) in a mortar with some sand; then follow the above procedure. Waxy leaves do not work well. The carotenoids are present in the leaf during its entire life span, so a green leaf from a maple tree or euonymus shrub, also known as burning bush, known to turn bright red in the fall, will show lycopene even when the leaf is green. In the fall, the chlorophyll decomposes before the carotenoids, so the leaves appear in a variety of orange and red hues. It is of interest to investigate the carotenoids of the tomato, of which there are some 80 varieties. The orange-colored tangerine tomato contains an isomer of lycopene. If a hexane solution of the prolycopene from this tomato is treated with a drop of a very dilute solution of iodine in hexane and then exposed to bright light, the solution will turn deep-orange in color, indicating that a cis-double bond has isomerized to the trans form. The product is, however, still not identical to natural lycopene.

Isomerization Prepare a hexane solution of either carotene or lycopene, and save a drop for TLC. Treat the solution with a very dilute solution of iodine in hexane, expose the resulting mixture to strong light for a few minutes, and then carry out TLC on the resulting solution. Also compare the visible spectrum before and after isomerization. Iodine serves as a catalyst for the light-catalyzed isomerization of some of the trans-double bonds to an equilibrium mixture containing cis-isomers.



Macroscale and Microscale Organic Experiments SIXTH EDITION Macroscale and Microscale Organic Experiments, Sixth Edition

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