Experiment 10 Isolation and Identification of the Major Constituent of Clove Oil

Steam Distillation, InfraredSpectrometry. Isolation andIdentification of the MajorConstituent of Clove Oil

Isolation ofNatural Products. Infrared Spectrometry.

Operations:

OP-15 Steam DistillationOP-34 Infrared SpectrometryOP-4 WeighingOP-13 ExtractionOP-14 EvaporationOP-20 Drying Liquids

Before You Begin

1 Read the experiment and read OP-15 and OP-34. Review the otheroperations as necessary.

2 Write an experimental plan for this experiment.

ScenarioA professional aromatherapist, Rose Otto, uses the essential oil from clovesas a treatment for toothache, muscle pain, ringworm, warts, flatulence, andgeneral exhaustion. But the latest batch of clove oil from her current sup­plier is darker than usual, has a harsh odor, and appears to be less effectivethan the oil she received previously. She suspects that the supplier has sub­stituted some clove leaf oil for true clove oil, which is distilled from clovebuds, the dried calyxes left after the flowers of the clove tree have fallen off.

Ms. Otto has asked you to provide her with an authentic sample offreshly distilled clove oil and tell her what's in it so that she can compare theauthentic clove oil with the product she has on hand. Your assignment is toisolate clove oil from ground cloves and identify its major constituent, whichis known to have the molecular formula ClOH120 2. Your supervisor believesyou can identify that constituent by using infrared spectrometry.

Applying Scientific MethodologyReading the experiment should yield a clue about the identity of the

unknown, which is one of those illustrated jn Figure 10.1 (p. 85). Then youcan develop a tentative hypothesis that will be tested when you obtain andinterpret the infrared spectrum. .

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EXPERIMENT 10

clove bud

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oII

CH2 = CHCHzS - SCHzCH = CHzallicin

Part I Mastering the Operations

Plants and Healing

As people seek alternatives to traditional medical practices, which empha­size the use of drugs and surgery to treat illness, various fields of alternativemedicine are gaining adherents around the world. These include aromather­apy, the use of essential oils to maintain health and treat illness; naturopa­thy, a system of treating diseases by using special diets, herbs, vitamins, andother natural healing methods; and homeopathy, which originally relied onthe use of minute doses of drugs to cure illness but now utilizes carefullyformulated mixtures of herbal medicines. Although some alternative med­ical practices may be associated with scientifically questionable theories,such as the idea (still accepted by some homeopathic practitioners) that thepotency of a drug increases with dilution, many fields of alternative medi­cine utilize plant-based medicines with a long history of healing efficacy.

In our age of modern medical miracles, people often associate herbalmedicine-the use of plants to treat and prevent illness-with witch doc­tors, shamans, or far-out medical cults. But herbal remedies have gainedpopularity in recent years as more and more people turn to echinaceae,goldenseal, and even garlic to help them stay healthy and cope with illness.Europe is well ahead of the United States in conducting scientific researchon herbal medicines. In fact, the popularity of six of the ten top-selling herbsin the United States has resulted mainly from European research. Forexample, a scientific team at the University of Dusseldorf, Germany,recently studied the active principals and biological effects of the purpleconeflower, Echinacea purpurea, which is used to treat colds and flu by stim­ulating the immune system. One objective of their research was to improvethe standardization of echinaceae extracts, helping to ensure that each doseprovides the same physiological activity.

After echinaceae, the most widely used herbal remedy in America isgarlic-one of the few remedies you are more likely to find in a grocerystore than a drugstore. Although researchers disagree on the virtues of gar­lic, there is evidence that it lowers cholesterol and triglyceride levels inblood, helps prevent blood clots that could lead to heart attacks or strokes,and lowers blood pressure. The main active ingredient in garlic is a sulfurcompound called allicin, which also gives garlic its distinctive smell.Unfortunately, cooked garlic contains little if any allicin, so many of thealleged benefits of garlic are obtained only from the raw'cloves or garliccapsules. Eating lots of raw garlic could have a negative impact on yoursocial life, but that may be a small price to pay for good health!

Other popular herbal medicines include goldenseal root, used for treat­ing peptic ulcers, infected gums, sore throats, skin infections, and variousother conditions; saw palmetto berries for treating nonmalignant prostatedisease; gingko leaf to improve blood flow in capillaries and arteries; aloevera gel to heal burns, cuts, and wounds; ephedra stems to treat asthma andhay fever; and ginseng root to enhance one's general well-being and revital­ize those weakened by old age or illness.

It should not surprise us that natural medicines can be effective. Thereare, after all, far more molecules in the world's natural life forms than havebeen synthesize'd in all the world's pharmaceutical laboratories. Many nat­ural molecules are alreac;iy known to have medicinal properties, and theremust be at least as marty more whose properties are yet to be discovered.

Experiment 10 Isolation and Identification of the Major Constituent of Clove Oil

Most of the drugs now prescribed by physicians were either derived fromnatural sources or developed by modifying the molecular structures of nat­ural substances. For example, the heart stimulant digitalis is extracted fromthe foxglove plant, and the molecular structure of aspirin (acetylsalicylicacid) is based on natural salicylates such as salicin from willow bark, whichhas been used for centuries by Native Americans to treat fevers.

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The six-carbon substituent in salicin is aglucose unit.

salicin

COzH

&OCOCH3aspmn

Another herbal medicine that was known to native healers long beforeits discovery by modern medicine is rauwolfia, a tranquilizer and antihyper­tensive drug derived from the East Indian snakeroot, Rauwolfia serpentina.The snakeroot plant has been used for at least 3000 years as an antidote forsnakebite, a treatment for insanity and stomach ache, and to soothe grumpybabies. Rauwolfia came to the attention of medical science when it wasfound to reduce blood pressure and cure some kinds of insomnia. Whenphysicians reported that heart patients treated with rauwolfia acted as ifthey didn't have a worry in the world, psychiatrists began using it to treatmentally disturbed patients. Analysis of the plant led to the discovery andcharacterization of its most active component, reserpine. No commerciallyfeasible process for synthesizing reserpine has yet been developed, so thisdrug is still derived from snakeroot extracts.

reserpine

A number of plants used for centuries by native healers and ancientphysicians have not yet been discovered by modern pharmaceutical compa­nies. Legend has it that Achilles, during the seige of Troy, used yarrow totreat the wounded Greeks. Its botanical name, Achillea millefolium, recog­nizes that tradition. The bruised leaves of yarrow help to stop bleeding, healcuts, and relieve the pain of a wound, so the plant has been used in medicalemergencies by backpackers and other outdoor adventurers. St. lohnswort(Hypericum perforatum)-was supposedly used by the ancients to drive awayevil spirits; today it is touted as an alternative to Prozac and similar drugs for

84 Part I Mastering the Operations

treating mild to moderate depression. The indigineous North Americanweed boneset (Eupatorium perfoliatum) provides a bitter tea that was afavorite Native American remedy for fevers and other ailments. The closelyrelated Joe Pye weed (Eupatorium purpureum) was named after a NativeAmerican who gained fame for using it to cure typhus. It is an effectivediuretic for the treatment of kidney and bladder ailments.

Although herbal medicines are generally milder and have fewer sideeffects than traditional prescription drugs, they are not all harmless.According to the U.S. Food and Drug Administration, herbal preparationscontaining ephedrine (also known as Ma huang) can cause heart attacks,strokes, seizures, and even death if used improperly. Unlike most prescrip­tion drugs, different preparations containing the same herb may varywidely in potency and physiological effect. But when used responsibly bywell-informed individuals, herbal medicine may provide a viable alterna­tive to the use of traditional drugs for treating illness and maintaininggood health.

Understanding the ExperimentThe essential oil of a plant is a mixture of volatile, water-insoluble compo­nents that exhibits the odor and other characteristics of the plant. In thisexperiment you will isolate the essential oil from cloves, which are obtainedfrom a small evergreen tree (Syzygium aromaticum) that grows in placessuch as Indonesia, Madagascar, and Zanzibar. Cloves contain about 16 per­cent by mass of the essential oil, a pale yellow liquid with a sweet, spicyaroma. Clove oil is unusual among essential oils in having only one majorcomponent, which comprises about 85 percent of the oil.

Because ground cloves lose their volatile components rapidly if leftstanding, it is best to grind fresh whole cloves just before using. Clovescan be ground quickly in an electric spice grinder, but a mortar and pestlewill suffice. Essential oils are nearly always isolated by steam distillation,in which steam forced through the plant material vaporizes the essentialoil, which is then condensed into a receiver along with water from thecondensed steam. This process is preferable to ordinary distillationbecause the volatile components distill at temperatures below their nor­mal boiling points, reducing or preventing decomposition due to over­heating. During the steam distillation, the presence of clove oil in thedistillate will be indicated by oily droplets or cloudiness. When all theclove oil has distilled, the emerging distillate should be as clear as waterand essentially odorless. Clove oil is separated from the distillate byextraction with dichloromethane (methylene chloride), and its majorcomponent is then separated from minor components by extraction withaqueous sodium hydroxide.

The major component of clove oil is a strong-smelling liquid that hasthe molecular formula ClOH 120 2•The structures of some natural compoundsthat have this formula are shown in Figure 10.1. Because these compoundshave different sets of functional groups, it is possible to distinguish them byusing infrared spectrometry. By observing the presence or absence of IRabsorption bands corresponding to specific functional groups, you should be

Experiment 10 Isolation and Identification of the Major Constituent of Clove Oil 85

able to arrive at the correct structure for the major component. The section"Interpretation of Infrared Spectra" in OP-34 describes the characteristicbands of organic compounds that have these functional groups.

aIICOH

o

°

xx°

aOH

Figure 10.1 Compounds with the molec­ular formula C lOH120 2

Directions

Safety Notes

Isolation of Clove Oil. Weigh about 5.0 g of fresh whole cloves and use aspice grinder or a mortar and pestle to grind them to a fine powder. Set upan apparatus for steam distillation [OP-15] using a large (250-500 mL) boil­ing flask and a steam trap, and have your instructor check your apparatus.Combine the ground cloves with 50 mL of water in the boiling flask, thensteam distill the mixture to extract the clove oil. Continue the distillationuntil a drop or two of th.e emerging distillate, collected on a watch glass, isOdorless and water-clear, with no oily droplets. You may need to distill

dichloromethane

Observe and Note: How does theappearance of the distillate changeduring the distillation?

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Take Care! Turn off the steam be­fore you remove the inlet tube fromthe apparatus.

Take Care! Avoid contact with di­chloromethane and do not breatheits vapors.

Stop and Think: What does this sep­aration procedure suggest about thenature of the major component?

Part I Mastering the Operations

150 mL of liquid or more before the distillate becomes completely clear. Besure to vent the steam line or raise the steam inlet tube above the liquidlevel in the boiling flask before you turn off the steam.

Extract the clove oil from the distillate [OP-13] with two 20-mL por­tions of dichloromethane and combine the extracts. (Waste Disposal: Theclove residue should be filtered through glass wool and placed in a solidwaste container. The water from the boiling flask and distillate can bepoured down the sink.)

Separation of Minor Components. Extract the active component ofclove oil from the dichloromethane solution with two 15-mL portions of1 M aqueous sodium hydroxide and combine the aqueous layers. (WasteDisposal: Place the dichloromethane in a chlorinated solvent recovery con­tainer.) Acidify the aqueous solution to blue litmus paper with 10 mLor more of 3 M hydrochloric acid. Extract this aqueous solution with two15-mL portions of dichloromethane and combine the extracts.

Isolation and Analysis of the Major Component. Dry the resultingdichloromethane solution [OP-20] with anhydrous magnesium sulfate.Evaporate the dichloromethane [OP-14] under vacuum using a cold trapuntil the boiling stops and the volume of the residue remains constant.(Waste Disposal: Place the recovered dichloromethane in a chlorinatedsolvent recovery container.) Weigh the liquid residue [OP-4] in an opencontainer, then reweigh it a minute or so later. If its mass decreases signifi­cantly between weighings, the evaporation should be resumed. Record theinfrared spectrum [OP-34] of the liquid.

Report. State the problem and your initial hypothesis. Identify as manybands in the IR spectrum as you can, and summarize and interpret this andother evidence. Deduce the structure of the active component of clove oil.Tell whether your hypothesis changed when it was tested and if so, why.State your final conclusion and tell how it is supported by the evidence.Calculate the percentage of the active component based on the mass ofcloves you started with. Turn in your product and IR spectrum with yourreport.

Exercises

1 Derive a systematic name for the active component of clove oil, anduse this to find its common name in The Merck Index or another ref­erence book.

2 (a) What property of the active component of clove oil made possi­ble its separation from the other components by the extractionprocess you used? Is this consistent'with the structure you chose forit? Explain. (b) Write equations for the chemical reactions involvedin the extraction and the subsequent acidification of the extract.

3 (a) Clove oil contains about 10% of a minor component that has theformula C12H140 3, which can be hydrolyzed to yield the major com­ponent and acetic acid. Deduce the structure of the minor compo­nent. (b) The percentage of the major component of clove oil

Experiment 10 Isolation and Identification of the Major Constituent of Clove Oil

actually increases as the cloves are steam distilled. Explain why andgive an equation for the reaction involved.

4 Describe and explain the possible effect on your results of the fol­lowing experimental errors or variations. (a) You stop the steam dis­tillation after collecting 75 mL of distillate. (b) You omit theextraction of minor components (see Exercise 3). (c) You don't evap­orate the dichloromethane long enough.

5 (a) Clove oil also contains a small amount of a substance whose system­atic name is (E)-4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4­ene. Write the structure of this compound and find its common name inThe Merck Index or another reference book.

6 Construct a flow diagram for this experiment, showing how the activecomponent is separated from the solid part of the cloves and the com­ponent mentioned in Exercise 3.

Other Things You Can Do(Starred projects require your instructor's permission.)

*1 Omit the Separation of Minor Components step in this experimentand analyze the clove oil by gas chromatography to determine theapproximate percentage of each component. You can also comparethe infrared spectrum of clove oil with that of its active component.

*2 Obtain and analyze an essential oil from orange peel as described inMinilab 10.

*3 Steam distill the essential oils from anise seed, caraway seed, orcumin seed following the procedure in this experiment but omittingthe Separation of Minor Components step. Each of these essentialoils contains a single major component that can be characterized byinfrared spectrometry.

4 Write a research paper about herbal medicine after referring tosources cited in the Bibliography.

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