Limonene, a compound with a familiar citrus odor, is found in the peel of many citrus fruits.
The structure of limonene, an unsaturated hydrocarbon, is shown below. You will note that, in addition to the unsaturation, limonene has a chiral carbon. When extracted from a biological source it should be optically active. In this exercise you will isolate (+)-limonene from orange peel using a form of steam distillation, and then test it for unsaturation and optical activity.
CH
3
H
3
C CH
2
Limonene (B. P. 178°C)
Normally, a liquid boils when its vapor pressure, which increases as temperature goes up, becomes equal to the surrounding pressure. Under ordinary circumstances, surrounding pressure is atmospheric pressure (about one atmosphere, or 760 mm of mercury at sea level, but about 730 mm of mercury here in Mesa). A solution (homogeneous mixture) will boil when the combined vapor pressures of its dissolved components is equal to the surrounding pressure. In the case of a solution, the pressure of each component is related to its concentration in the mixture. The boiling point of a homogeneous mixture is usually between the boiling points of its individual components. A heterogeneous mixture , on the other hand, will also boil when the combined vapor pressures of its components equals the surrounding pressure. The vapor pressures of the individual components of a heterogeneous mixture are independent of one another and unrelated to their concentrations. The total vapor pressure of a heterogeneous mixture is shown in the following equation:
P° total
= P°
A
+ P°
B where P° total
is the total pressure of a system at a given temperature and P°
A
and P°
B
are the individual pressures of components A and B at the given temperature. Generally, a heterogeneous mixture will boil at some temperature below the boiling point of either component alone. This effect can be seen by examining Figure 1, where the individual vapor pressures of two liquids are plotted against temperature. Note that in the figure, compound B, which has the lowest boiling point, will boil at a temperature slightly above 125°C. When compound A is also present, the combined pressures are such that the mixture will boil at about 85°C. This boiling temperature is lower than the boiling point of either component alone and it is due to the combined effects of both compounds.
Isolation of (+)-Limonene from Orange Peel, Page 1

t.
The vapor pressure curves of two immiscible liquids. Water has a vapor pressure of 647.1 torr at
96 °C. The second liquid has a vapor pressure of 88.59 torr. Since the sum is 735.7, it is not enough to boil at sea level (760 torr, on average), but is enough to boil here in Mesa, at 1205 ft above sea level where the atmospheric pressure is usually around 730 torr.
The effect described above is put to use in a technique called steam distillation, where an organic compound of moderate volatility (and moderate vapor pressure) is distilled as part of a heterogenous mixture with water. The boiling point of the mixture is slightly lower than 100 °C (the boiling point of water). At this temperature, a fraction of the condensate (the liquid collected from the condenser) will be the compound of interest. The greater the vapor pressure of the organic compound, the larger the fraction that will co-distill with water. This technique is considerably more gentle than common distillation, where the compound of interest may decompose at temperatures approaching its own higher boiling point.
Peel three large or four moderate size oranges (or other citrus) with a vegetable peeler. Do your best to remove just the exterior, orange portion of the peel. The skin peelings will be like carrot peelings. The white pulp contains little or no limonene. Collect the peelings and put them into a blender. Add about 150 mL of deionized water and turn on the blender. Blend the mixture until a fine, smooth puree is obtained. Pour the mixture, along with a few boiling stones, into a 500 mL round-bottom flask. Connect the flask to the steam distillation system shown in Figure 2
.
Isolation of (+)-Limonene from Orange Peel, Page 2

Figure 2: Steam Distillation Apparatus
Using a heating mantle , heat the round-bottom flask to boiling. A mixture of water and limonene will evaporate and then condense in the condenser and drip into the collection flask. Be careful not to boil the mixture too vigorously, or the crude mixture may bump into the condenser and contaminate the condensate. If this happens, you will need to clean the system and begin the process again. Periodically add 15 to 20 mL increments of water to the boiling flask from the separatory funnel (in this case used as an addition funnel) to replace the water that has been distilled.
Collect about 150 mL of condensate in the collection flask. If the steam distillation worked properly, an upper, organic film of limonene should be visible in the collection flask. If necessary, replace the collection flask and collect more liquid to improve your yield of limonene.
Transfer the condensate to a separatory funnel. Discard the lower, aqueous layer, and collect the upper limonene layer in a pre-weighed vial. Weigh the vial with the limonene oil. After that, add a few crystals of anhydrous calcium chloride or other drying agent. Record this as a mass of the wet limonene oil. Allow the limonene to dry over the drying agent until the next lab period.
After your limonene sample has dried, carry out the following procedures and tests to confirm its identity
1. To separate the oil from the drying agent, filter the oil through a Pasteur pipette equipped with a cotton plug. Collect the oil in a clean pre-weighed vial and record its mass.
2.
Dissolve two or three drops of dried limonene in 0.5 mL of dichloromethane. To this solution, add one or two drops of bromine test reagent. Rapid decolorization of the bromine is characteristic for double bonds. Record your observations. A test with KMnO
4
could also be used here, where a color change from purple to brown would also indicate the presence of a double bond.
3.
Take an IR spectrum of your sample. Compare your spectrum to that of authentic limonene shown in Figure 3.
Isolation of (+)-Limonene from Orange Peel, Page 3

4.
Make up a solution of 1.0 g limonene in 25 mL absolute ethanol. Put this solution in a 1 dm
(10 cm) polarimeter tube and measure the optical rotation of the solution. You may need to pool samples from several groups to perform this measurement. Your instructor will explain how to use this instrument. Since this test may require you to use your entire sample, you should perform this experiment last. After you have taken this measurement, calculate and record the specific rotation of your compound by using the formula below:
 t


 c
 
Where:
 t

is the specific rotation at temperature t and wavelength λ;
 is the observed rotation, c is the concentration of the sample in g/mL,  is the light path in dm.
5.
The reported specific rotation for neat (pure, undiluted) (+)-limonene is 125.6° at 20°C using the
D line from a sodium vapor light (a specific wavelength of yellow-orange light.)
Figure 3: IR Spectrum of Limonene courtesy of SDBSWeb : http://www.aist.go.jp/RIODB/SDBS/
(National Institute of Advanced Industrial Science and Technology, July 22, 2006)
Isolation of (+)-Limonene from Orange Peel, Page 4

Name ____________________________________
1.Bromine decolorization test result
2. Based on the bromine test, how would you classify limonene?
3. Based on your infrared spectrum (attach to this report) how does your compound compare to authentic limonene?
4. Based on your polarimeter measurement, what is the specific rotation of your compound? (Show calculations.)
Mass of the wet oil _____________________________ g
Mass of the dry oil _____________________________ g
Mass of water removed _________________________ g
1.
Based on the relative amounts of water and limonene collected in this experiment, how does the vapor pressure of limonene compare to that of water?
2.
What type of compound, based on its physical properties, can be steam distilled?
Isolation of (+)-Limonene from Orange Peel, Page 5

3.
Draw the perspective drawing of the two limonene enantiomers (the racemic mixture is actually called cinene). Label the two enantiomers with the correct R and S designation.
4.
Write equations representing the reactions of limonene with bromine and with cold, basic
KMnO
4
. These need not be balanced, but should show the organic products with correct stereochemistry
5.
Should it be possible, using properties other than optical rotation, to distinguish (+)-limonene from its enantiomer?
6.
If you were the first to isolate a new optically active compound like limonene and observed that its rotation, at a certain dilution, was +100°, what experiment would you suggest to demonstrate that its rotation was not actually -260°?
Isolation of (+)-Limonene from Orange Peel, Page 6