Macroscale and

Microscale
Organic
Experiments
SIXTH EDITION

Kenneth L. Williamson
Mount Holyoke College, Emeritus

Katherine M. Masters
Pennsylvania State University

Australia Brazil Japan Korea Mexico Singapore Spain United Kingdom United States

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 Macroscale and Microscale
Organic Experiments, Sixth Edition
Kenneth L. Williamson, Katherine M. Masters
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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.
Isoprene
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Chapter 9 Column Chromatography 201
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
Lycopene, the red pigment in tomatoes, is a C40-carotenoid made up of eight five-
carbon isoprene units. -Carotene, the yellow pigment of the carrot, is an isomer of
lycopene in which the double bonds at C1C2 and C'1C'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 -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 reac-
tions. 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 contain-
ing 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.
202 Macroscale and Microscale Organic Experiments

Carotenoids are highly sensitive to photochemical air oxidation; therefore, pro-

tect solutions and solids from undue exposure to light and heat and work as rap-
idly 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 sol-
vents 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 evapo-
rated at room temperature under vacuum to leave the crude carotenoids.

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 Bchner funnel. Scrape out the
tube with a spatula, let it drain thoroughly, and squeeze out as much liquid as pos-
sible 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 vigor-
ously. Filter the mixture on a Bchner 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 cal-
cium chloride pellets, filter the solution into a small flask, and evaporate the solu-
tion 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,
w never heating the sample above 50C. Determine the weight of the crude material.
Video: Column
Chromatography; Photo: Column It will be very small. If the residue is dry, as it should be, add just enough
Chromatography 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 evap-
orate the mixture to dryness, again without heat.

Column Chromatography
The crude carotenoid is to be chromatographed on an 8-cm column of basic or neu-
tral 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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 Chapter 9 Column Chromatography 203

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% ace-
tone 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 com-
bine your purified products with those of several other students, evaporate the
solution to dryness, dissolve the residue in deuterochloroform, CDCl3, and deter-
mine 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 manu-
factured 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 pur-
pose. Used plant material and dry TLC plates can be discarded in the nonhaz-
ardous waste container.

For Further Investigation

The carotenoids of any leaf can be isolated in the manner described in this exper-
iment. 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.

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