LIMONENE FROM CITRUS FRUIT
Pre-lab:
Read chapters 4, 9, 18, 18, 20 in Zubrick for the first week’s work, and chapters 10, 11, and 15 for the second. You should also consult the stereochemistry chapter in Wade (see the lecture syllabus for the assigned sections).
Your pre-lab should consist of a title, a brief objective, and a table
of physical properties (including hazards) for limonene and methyl tert-butyl
ether (MTBE), especially the specific rotations for the enantiomers of
limonene.
BACKGROUND:
Many natural products (naturally occuring compounds such as plant metabolites) have been discovered which have useful commercial or medicinal applications. For example willow bark contains salicylic acid which has medicinal value as an antipyretic, analgesic, and antirheumatic drug. Some natural products, such as the limonene you will be isolating in this experiment, are present in such large concentrations that the natural source meets all needs. Unfortunately, the compound of interest is often present in too low a concentration in the natural source to be of widespread use. One example is taxol, a compound with anti-cancer activity which is present in the bark of the pacific yew. To solve this problem chemists will isolate the biologically active ingredient, determine its structure, and develop a practical synthesis of the compound. Often, especially in medicinal chemistry, structural analogues of the naturally occuring compound will also be synthesized, in the hopes of maximizing the beneficial effects and minimizing unwanted side-effects.
The citrus oils that you will isolate have commercial use as fragrances and food flavorings. Limonene is the major component (~ 90%) of the essential oil obtained from citrus fruit peel and is extracted for use in perfumes, food flavorings, shampoos, etc. Limonene and pinene, another component of citrus oils, belong to a class of compounds known as terpenes, specifically the monoterpenes. Terpenes are composed mostly of hydrogen and carbon and are dimers of two 5-carbon isoprene units. The basic carbon skeleton of an isoprene unit is given below. For further information on terpenes you may consult section 25-8 in Wade.
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Limonene is a chiral molecule, and both enantiomers are found in nature. However, while both enantiomeric a -pinenes are synthesized by citrus trees, only one enantiomer of limonene is synthesized selectively. Your goal is to determine which one it is: the R-(+)or the S-(-). Enantiomers are indistinguishable in their chemical and physical properties unless another chiral reagent is employed. Melting points, boiling points, densities, refractive indices, solubilities, etc. are identical. However, enantiomers do exhibit different optical activity. Polarized light is a chiral reagent and can distinguish between enantiomers. They rotate polarized light with equal magnitude but in opposite directions. You will measure the optical rotation of the oil you isolate, and determine which enantiomer of limonene it contains. The human body is also chiral, and while it is difficult in the lab to distinguish between enantiomers, our senses of smell and taste can often do so readily. For example, the two enantiomers of carvone, another terpene, have markedly different odors. The (+)- carvone found in caraway seeds gives rye bread its flavor, while (-)-carvone is found in spearmint.
SAFETY:
Week #1: isolation of citrus oil by steam distillation
Choose your citrus fruit: 1 orange or 1 lemon.
Peel your fruit and break the rind into small pieces so that you can fit them through the neck of a 250-ml round-bottom flask (RBF). Your metal spatula can be used to cut the rind. Add three boiling chips and 100 ml of distilled water to the RBF. Complete the set-up for the distillation (see the illustration on page 146 of Zubrick) as demonstrated by your lab instructor. Use your 25 ml graduated cylinder as the receiving flask, sit it in an ice/water bath in a 400-ml beaker, and wrap its neck with Al foil to minimize vapor leakage. Set the variac to 70 volts. Remember that the heating mantle is plugged into the variac, and the variac is plugged into the electrical outlet. Have your instructor inspect your set-up before boiling commences.
Collect at least 50-ml of distillate (keep a record of the distillation temperature) and store it in your labelled and stoppered 125-ml standard-taper (TS) Erlenmeyer flask (EF). When the distillation is over lower the heating mantle to let the distillation flask cool off. When you are finished collecting distillate wrap parafilm around the stopper of your EF and store the flask in your locker. Disassemble the apparatus working backwards from the receving end. Clean your glassware first by wiping the grease off the joints, then with soap and water followed by an acetone rinse. Acetone rinses go in the waste bottles located in the hood. Pour out any water remaining in the distillation flask, and dispose of the citrus peel in the wastebasket.
Week #2: further isolation and characterization of citrus oil
Get a 125 ml separatory funnel and 10 ml volumetric flask from the bins in the hood. There are separatory funnel racks in the cabinets under the sinks.
Transfer the distillate to the separatory funnel. Add 10 ml of MTBE to the Erlenmeyer flask and rinse the contents into the sep. funnel. Stopper and shake the sep. funnel, venting occasionally, to EXTRACT the citrus oil. Let the layers separate. Figure out which layer is which, and transfer the organic layer to a clean and dry 125 ml Erlenmeyer flask. Return the water layer to the sep. funnel and repeat the extraction two more times, each time using a fresh 10 ml portion of MTBE. Add each MTBE extract to the same Erlenmeyer flask. DRY the combined organic layer by adding MgSO4 until fresh powder no longer clumps. Allow the drying agent to work for ~10 minutes.
GRAVITY FILTER the dried organic layer into your PRE-WEIGHED 50 ml pear-shaped flask (PSF), use a little more MTBE to rinse the drying agent, and use the ROTARY EVAPORATOR to evaporate off the MTBE. Continue until you reach a constant mass. The residue is the desired citrus oil. Note its fragrance and determine its MASS.
For the determination of OPTICAL ROTATION you must prepare a solution of your entire sample in 95% ethanol. Using a disposable pipette, transfer your oil to the 10 ml volumetric flask. Some ethanol can be used to rinse the PSF. Swirl the volumetric to dissolve your oil in the ethanol, fill the flask to its mark with the ethanol, stopper, and shake to make sample uniform. Obtain a polarimeter tube and using another clean pipette fill the tube with your solution (watch for air bubbles). Proceed to the polarimeter where the instructor will demonstrate its use. When you have measured the observed rotation of your solution, pour the ethanol/oil solution into the appropriate waste bottle in the hood and rinse out the polarimeter tube with a little ethanol. The aqueous layer from the extraction can be discarded down the sink.
Calculate your specfic rotation from the experimentally measured optical
rotation, cell length (1 dm) and the concentration of your sample in g/ml
(see page 190 in Wade).
Report:
The report for this experiment will consist of abstract, results, and conclusions sections, and is due in lab the week of Sept. 24.