EXPT 115

STEAM DISTILLATION OF A SPICEMost introductory information taken directly from: William H. Miles, and Patricia M.

Smiley, J. Chem. Ed. 2002 79, 90. Revised 10/17/06

PRELAB EXERCISE

Besides being bacteriostatic or antibacterial, essential oils of spices can display othertypes of bioactivity. Give the name of an oil and its bioactivity (besides beingbacteriostatic or antibacterial). If using the Internet, please include the website reference.

INTRODUCTION

Chemistry and MedicineSince ancient times, humankind has sought medicines from nature. Quinine, theantimalarial compound isolated from the bark of the cinchona tree, and reserpine, theantidepressant from the Indian plant Rauwolfia serpentina, represent two of the morefamous natural products used in modern medicine.

Today, pharmaceutical companies use several approaches for the discovery of newcompounds to treat human illness, including the screening of natural products derivedfrom bacteria, fungi, plants, or animals (not puppies or even mice, but animals like slugsor sponges, simple multi-cellular, bottom-dwelling animals called “Porifera”). Forexample, extraction of a marine sponge with an organic solvent can yield a complexmixture of organic compounds, which can then be tested for biological activity or are“bioassayed”. If the mixture shows promising biological activity, the components of thecomplex mixture may be separated and purified. When further testing identifies theactive component, its chemical structure can be determined using modern spectroscopictechniques (IR, NMR, Mass Spectrometry, X-ray crystallography). Even if the newcompound has side effects that render it unsuitable as a drug candidate, thepharmaceutical chemists will use the compound as a guide for the synthesis of new drugcandidates with similar structures.

The synthesis of organic compounds, whether of natural products or of "unnaturalproducts" (compounds that are not found in nature), has been a primary goal of organicchemists. Some of the first synthetic compounds produced industrially were dyes,fragrances and flavorings. It was not until the twentieth century that synthetic drugs weresuccessfully developed. The synthesis of the antisyphilitic drug arsphenamine,Compound 606, by Ehrlich in 1910, was a dramatic demonstration of the power ofchemistry to attack a medical problem. (Yes, arsphenamine contains arsenic. Not goodstuff, but remember, you’re dying of syphilis, so you can’t be fussy.) Since then, manynew compounds, including biologically active compounds found in nature, have beensynthesized in the laboratory and have become useful drugs. For example, Ritalin is usedfor the treatment of attention deficit disorder (ADD), 5-fluorouracil is a potent anti-cancer drug, and L-DOPA is used to treat Parkinson's disease.

Essential oils, which are isolated by steam distillation of volatile organic compoundspresent in plants and animals, have a long and rich history in medicine. Typically,complex mixtures of organic compounds, essential oils, have been used to treat a varietyof illnesses for thousands of years. In some cases these essential oils do have therapeuticutility by modern medical standards. The essential oils of cloves and thyme containphenolic compounds (organic molecules that have an OH group attached to an aromaticring), which are effective antiseptics (germ-killers). Even before the establishment of thegerm theory, the antiseptic power of both thyme oil and clove oil was recognized. JosephLister, whose name is immortalized by the mouthwash Listerine, used synthetic phenol(carbolic acid) to prevent infection during surgical procedures in the mid-nineteenthcentury, revolutionizing the medical treatment of wounds. He recognized that antisepticchemicals can prevent infection and proved the value of some of the old herbal remedies.Phenolic compounds, both synthetic phenol and thyme oil, were used as antiseptics untilWorld War I.

The clove tree produces flower buds which, when harvested and dried, become thefamiliar spice of the same name. It has long been known that cloves contain an ingredient

with an anesthetic property that is particularly effective against dental pain. Beforemodern medicine developed better remedies, people often chewed on cloves to dull thepain of a toothache. The active compound responsible for this effect is found in theessential oil of cloves which makes up approximately 16-18% of the clove by weight.The major component of this oil is the compound eugenol. Eugenol has a boiling pointof 254° C. It would be difficult to isolate this oil by simple distillation since this hightemperature could lead to its decomposition as well as the decomposition of othercomponents of the clove. However, eugenol can be isolated relatively easily from clovesusing the technique of steam distillation. Once the aqueous distillate containing the cloveoil has been obtained, the oil can be separated from the water by extraction into theorganic solvent dichloromethane.

The dried fruit of cumin (Cuminum cynimum L.), a small shrub cultivated in easternEurope and India, is an important seasoning in curries, goulashes, and sausages. It is alsoa major component of chili powder used in Mexican food. The major volatile constituentof cumin is p-isopropylbenzaldehyde (cuminaldehyde). Cumin oil also containslimonene and other compounds which contribute to the aroma of this condiment.

The Isolation of Organic CompoundsAs you know, functional groups give the molecules distinctive chemical reactivity, aswell as play a role in the physical properties of compounds. Different physical propertiesallow the separation of one component from a mixture. In some cases, separations can bebased on the solubility of the compound in a given organic solvent, so that a compoundcan be recrystallized, as you learned in the recrystallization technique experiment. Asyou will learn in this technique experiment, organic compounds with appreciable vaporpressure can be separated by distillation at atmospheric pressure, or if their vaporpressure is low, steam distillation or vacuum distillation at reduced pressure can be used.In some cases, as in the Liquid/Liquid Extraction Technique Experiment, separations canbe done based on the acid/base chemistry of the organic molecules. Many separations arebased on chromatographic techniques, which you’ll study later.

Biological TestingThe testing of compounds for biological activity is the first step in the determination oftheir potential as a drug in medicine. In most cases, the compound is tested against anorganism (bacteria, fungi, or virus) in vitro, that is, outside a living organism in acontrolled environment such as a Petri dish. One method for testing antibiotic activity isto apply the compound onto a sterile paper disk and place the disk on an agar plate

containing a culture of bacteria. If the compound is active, then a zone of inhibition willappear around the disk; other areas of the Petri dish will show evidence of bacterialgrowth. The size of the zone indicates the potency of the drug. Adjusting the amount ofcompound applied to the disk allows a crude dosage effect of the compound to be seen.If the pharmaceutical companies discover a promising lead, the next step is to investigatethe potency of the drug in vivo. Animal studies determine the effectiveness of the drug aswell as provide information about the toxicity, metabolic pathway of decomposition, andside effects of the drug. After exhaustive animal studies, the drug will then undergohuman trials. Human trials start with Phase I clinical trials, in which the toxicity anddosage response of the drug is determined with healthy volunteers. In Phase II clinicaltrials, the drug is tested on people (100-300) who have the disease. In Phase III clinicaltrials, the compatibility of the new drug with other medications is determined in severalclinics and hospitals with as many as a thousand patients. If no complications or seriousside effects are found, then the company can apply for Food and Drug Administration(FDA) approval for this drug in the treatment of a given disease. If FDA approval isgranted, then the drug is available for physicians to prescribe. Since problems mayappear even after such extensive testing, the pharmaceutical companies continue tomonitor the use of their drugs.This process is long and expensive. The average cost has been estimated at 250 milliondollars, with at least a ten-year span from the initial testing of the drug to final FDAapproval. It has been estimated that only one compound out of ten thousand compoundsprepared by a pharmaceutical company receives final FDA approval. Although theresearch and development costs are considerable in the pharmaceutical industry, therewards are significant; a blockbuster drug like Prozac has sales in the range of twobillion dollars a year.

Modeling the Drug Discovery ProcessThe separation of a biologically active compound such as eugenol from a mixture oforganic compounds obtained from a natural source (cloves in the case of eugenol), andbiological testing of this compound (as well as the other compounds) using a simplebioassay for antibiotic behavior, models many aspects of the drug discovery process. Youwill separate, isolate and test the antibiotic activity of the initial organic extract of thesteam distillate using Bacillus cereus growing on agar plates, which you will prepare.

Steam DistillationSteam distillation is another way to distill high boiling substances and is useful for theisolation of oils, waxes, and some complex fats. Any organic liquid that is immisciblewith water can be distilled at a temperature around 1000C, the boiling point of water. Forexample, eugenol boils at 254°C and would decompose extensively if we tried to distill itat this temperature. We could use vacuum distillation or steam distillation. How doessteam distillation allow the vaporization of such a high boiling substance as eugenol at ~100°C? Unlike the normal distillation of two miscible liquids, in steam distillation, eachcomponent of an immiscible liquid mixture contributes to the total vapor pressure as ifthe other component were not present. In other words, the total vapor pressure of themixture is the sum of the vapor pressure of the water plus the vapor pressures of each ofthe organic components in the mixture.

Ptotal = P0component 1 + P0

component 2 + P0component 3 + P0

component n + P0water

A liquid or liquid mixture boils when the total vapor pressure of the liquid is equal toatmospheric pressure. The vapor pressure of water is 760 torr at 100°C. The presence ofany immiscible organic material contributes to the total vapor pressure and thus causesthe boiling point of water to drop below 100°C. While eugenol only has a vapor pressureof about 20 Torr at 100°C, the eugenol vapor is swept out of the boiling flask by theboiling water vapor or steam into the condenser, separating into liquid water with smallamounts of immiscible eugenol. This yields a cloudy distillate of water and eugenol(plus other volatile organic substances). Since it takes a lot of water vapor to sweep outthe higher boiling organic oils, it is often necessary to add water to the distilling flask atfrequent intervals throughout the distillation until the organic compounds have beenremoved from the mixture.

Biological TestingEssential oils, which are isolated by steam distillation of volatile organic compoundspresent in plants and animals, have a long and rich history in medicine. Typically,complex mixtures of organic compounds, essential oils, have been used to treat a varietyof illnesses for thousands of years. In some cases, these essential oils have therapeuticutility by modern medical standards. The essential oils of cloves and thyme containphenolic compounds (organic molecules that have an OH group attached to an aromaticring), which are effective antiseptics (germ-killers). Even before the establishment of thegerm theory, the antiseptic power of both thyme oil and clove oil was recognized.

The testing of compounds for biological activity is the first step in the determination oftheir potential as a drug in medicine. In most cases, the compound is tested against anorganism (bacteria, fungi, or virus) in vitro, that is, outside a living organism in acontrolled environment such as a Petri dish. One method for testing antibiotic activity isto apply the compound onto a sterile paper disk and place the disk on an agar platecontaining a culture of bacteria. If the compound is active, then a zone of inhibition willappear around the disk; other areas of the Petri dish will show evidence of bacterialgrowth. The size of the zone indicates the potency of the drug. Adjusting the amount ofcompound applied to the disk allows a crude dosage effect of the compound to be seen.

This will be a two-day lab. On the first day you will follow procedure 1, steam distillationof a spice; you may not get to do the extraction on day one – if not, do it on day two. Onday two you will be following procedure 2, biological analysis. Before doing the steamdistillation procedure, please read the following article, whose link can be found on thecourse website, by W. H. Miles and P. M. Smiley printed in the Journal of ChemicalEducation (2002). You should also read “Antimicrobial Effects of Spices and Herbs” byO. Peter Snyder, 1997; use Google to find this article on the web.

PROCEDURE 1: STEAM DISTILLATION OF SPICES

Distillation Set-upAssemble the apparatus for steam distillation as shown in Figure 1. Start by placing yourmagnetic stirrer on the base of a ring stand. Attach the heating mantle (without sand init) to the ring stand so it rests on top of the stirrer. Plug the heating mantle into theVaristat, a variable voltage controller, on the side of the hood and plug the Varistat intothe wall socket. Place a 1” stirring bar in a dry, 100 mL round-bottomed flask (blue kit)and clamp the neck of this flask to the ring stand so that it fits snugly in the heatingmantle. Stirring promotes even boiling and avoids “bumping”. As you assemble theapparatus, lightly grease each ground joint by putting a very small dap of grease on theinner joint and spreading it over the surface of the joint (do not use excess grease as itwill contaminate the product). Equip the 100 mL round-bottomed flask with U-shapedadapter (blue kit), which is equipped with the distillation head (blue kit) and a separatoryfunnel. To this distillation head, equip a water condenser in the side joint and a stopperto the top joint. Again, be sure to lightly grease the joints; when pressed into the outerjoint and twisted, the connection should appear transparent. Equip the water condenserwith the water tubing from your locker; water-in on the “lower end” of condenser andwater-out on the “higher end” of the condenser. To the end of the condenser, attach thevacuum adapter (blue kit); be sure to use a rubber band to secure this attachment. Youcan use an Erlenmeyer flask or a 50 mL round bottom flask as the collection flask tocollect the distillate; cool this flask in a beaker of ice water. A thermometer isunnecessary as the temperature will always equal approximately the boiling point ofwater.

Figure 1. Set-up for steam distillation

Water enters the condenser at the connection nearest the distillation receiver. Because ofthe large heat capacity of water only a very slow flow is needed; too much water pressurewill cause the tubing to pop off, causing a flood. The condenser should be clamped to aseparate ring stand as shown above. A rubber band (Common Shelf) should be used tohold the angled vacuum adapter to the condenser.

Steam DistillationChoice a spice: tumeric, nutmeg, or cloves. Weight out 4 g of spice and grind up thespice using a mortar & pestle (found on the Common shelf). After grinding the spice,please wipe the mortar & pestle with a paper towel since these pieces will be shared.

Transfer the ground spice to the 100 mL flask using the weighing paper as a funnel. Add50 mL of distilled water to the 100 mL flask. Fill the separatory funnel half-way withdistilled water (be sure it’s closed!) Wrap the top of the 100 mL round bottom flask andthe 3-way connector with some glass wool to insulate it so that the steam vapors don’tcondense at that site. Start stirring and heating the mixture with a Varistat setting of 60.Lower the heating voltage if foaming is a problem. You should try to adjust the heatingso that distillate drips at a rate of one drop every 2-5 seconds. Begin adding the water tothe flask in small amounts via the separatory funnel so that it roughly matches the rate ofdistillate collecting in your collection flask. Replenish the ice in the beaker that is used to

cool your collection flask as necessary; use a Pasteur pipet to remove some of the waterbefore adding more ice. The distillate should be cloudy when the natural products are co-distilling with the water. When the distillate drops becomes clear you can remove theheat from the 100 mL round bottom flask.

Isolation of OilRemove the collection flask containing your essential oil and water. Use your 125 mLseparatory funnel to extract the essential oils in your distillate into dichloromethane.Making sure the stopcock is closed, transfer the contents of the collection flask to theempty 125 mL separatory funnel. Remember a stopcock can leak so always double checkyour stopcock to make sure it is tight and always have an Erlenmeyer flask or beakerunder the funnel to catch any possible leaks. Add 10 mL of dichloromethane (CH2Cl2) tothe separatory funnel and follow the extraction procedure you learned in the liquid/liquidextraction experiment. Repeat this process two more times with 10 mL additions of freshdichloromethane. Combine all organic layers in a 125-mL Erlenmeyer flask labeled“TOTAL OILS”. Dry the organic layer for 10 min over anhydrous sodium sulfate(Na2SO4). Extract can be decanted into a tared 25-mL Erlenmeyer flask labeled“TOTAL OILS”. Allow the dichloromethane to evaporate until the next lab period.Alternatively, if time permits one could blow off the dichloromethane with a stream ofnitrogen while immersing the flask in a beaker of warm water.

Gas Chromatography (GC) and GC-Mass Spectrometry (GC-MS) Analysis:You may be assigned to analyze your “total oils” mixture using the Hewlett Packard 5890GCs and the GC-MS. You will need to take your small vial containing thedichloromethane and the oil mixture to the instrument room for analysis. You shouldhave signed up for time on the instrument and you will need to do the CALIOPE GCtutorial on the computer adjacent to the GC. The tutorial is interactive so it will showyou how to the do the analysis, while you go through it. The tutorial will not provide youwith a temperature program so use the following: column temperature program from 40to 280°C at 10°/min. Inject 1 µL of solution. After obtaining your chromatogram, placeit (along with the GC sheet) in the designated bin near the instrument for approval by Dr.Bortiatynski. Dr. B will analyze the quality of the spectrum to ensure that the sample isready for GC-MS analysis. Approval turnaround time will be 1-2 days. Once seen byDr. B, your chromatogram will be place in a designated bin. Please look for hercomments before running the GC-MS. When you run the GC-MS, be sure to bring yourGC chromatogram along! See the inside back cover of the lab guide for samplepreparation instructions for both analyses.

Procedure 2: Bioassay of Spice Oil(s)

Essential oils extracted from natural sources may exhibit antibiotic behavior. Thisprocedure will test if your isolated oil has any bioactivity.

condenser

burette

You will use prepared sterile agar plates and inoculate them with with Bacillus cereus totest for its antibiotic behavior. Try to share an agar plate with a labmate. Each studentcan use one half of the plate.

It is very important that the following procedure is observed or contamination from otherbacteria will interfere with the results of the experiment. Whenever you handle the sterileagar petri dishes try to minimize any exposure to random forms of bacteria or dirt in thelaboratory. Always wear gloves that have been sprayed with ethanol when handling theplates and the cultures, because your hands can easily contaminate the agar. Open thecover of the dish a few inches vertically so that you have just enough room to swab theplate with a known strain of bacteria or insert a testing disk and immediately cover theagar to reduce the risk of outside contamination. You will use a pair of forceps to handlethe test disk and sterile swabs to streak the plates with the cultures. After preparing yourassay you will observe the growth of bacteria on the plate. If the bacteria do not grow inthe area surrounding the disk treated with your essential oil then one or more of thecompounds in this isolated mixture is exhibiting antibiotic behavior. See Figure 2 for anexample agar plate.

Working with BiohazardsYou will be working with bacterial cultures this semester to investigate the antibacterialactivity of spices and/or compounds that you make. The cultures chosen for thisexperiment are non-pathogenic, but the materials that come in contact with them must becleaned and or disposed of in a very specific manner. The following instructions are tobe used whenever handling bacterial cultures in the lab.

1. As with any experimental laboratory procedure, safety glasses must be worn at alltimes!

2. Wash your hands with antibacterial soap.3. Put on a new pair of disposable gloves and spray them lightly with ethanol.4. Prepare all bench surfaces for bacterial experiments by wiping them down with

either ethanol or antibacterial wipes or spray. Your TA will inform you of thelocation of these materials. All paper towels used in the preparation of benchspaces can be thrown in the regular garbage cans.

5. Line the clean workspace with a piece of clean bench paper, see TA for location.6. Assemble all glassware and laboratory materials listed in the instructions and

place them on the bench paper in the order in which they will be used.7. The materials that will come in contact with the bacteria must remain sterile until

inoculation, otherwise you will have interfering results from naturally-occurringbacteria. If any supplies are wrapped, do not remove the protective covering untilyou are ready to use them. You may also need to sterilize some equipment justprior to use and this can easily be accomplished by spraying or dipping these toolsin ethanol. All forceps used any bacterial experiments will be sterilized bydipping them in ethanol. Remember the ethanol must be allowed to evaporatefrom the surface of the tools, otherwise it will kill the desired bacteria.

8. Whenever you are transferring bacterial cultures it is important to keep anymaterials that are used in the process contained. Do not lie anything down thathas touched the bacteria. Anything that comes in contact with the bacteriamust be either sterilized or disposed of in the biohazard waste. All cottontipped applicators must be immediately placed in the orange waste containerslabeled biohazard. All forceps that come in contact with bacteria must besterilized by dipping them in alcohol.

9. Once the bacterial transfer is complete, the cultures are to be returned to the TA,and the bench paper is to be folded and placed in the biohazard waste.

10. Whenever you are handling agar plates inoculated with bacterial cultures, youmust be wearing disposable gloves.

11. All used agar plates must be disposed of in the biohazard waste.Whenever you are finished handling the bacterial cultures and any materials that havecome in contact with the cultures dispose of your gloves in the biohazard waste and washyour hands with hand sanitizer and then with antibacterial soap.

Procedure for the transfer of sterile cultures:All students must be wearing gloves during this entire procedure.

1. Obtain a prepared, sterile agar plate from a designated refrigerator.2. Label the nutrient agar plate with your last name, the name of the bacteria and the

name of the spice(s) that will be placed on the plate. The plate is to be dividedinto 4 equal quadrants. Each student will have two of these quadrants, one fortheir control and one for their spice. Use the marking pen to delineate thesequadrants and use this area as a guide preparing the plates. Be very careful notto remove the cover of the plate during this process.

3. Next, the bacteria (see stockroom) will be transferred to the plate using thefollowing steps:

a. Insert a sterile swab into the culture tube containing the bacteria.b. Remove the swab from the culture tube and then tilt the cover of the agar

plate at an angle to keep exposure to unwanted bacteria to a minimum. Donot lay the top of the plate on the lab table and do not allow anyone tobreath come in close contact with the plate.

c. The moist swab is brought in contact with the agar using a rubbingmotion. Start at the center of the plate and very gently rub back and forthmoving toward on edge of the plate. Turn plate 90 degrees and repeat therubbing of the swab. Turn the plate 135 degrees and rub the remainingsection of the plate. The plate should now be evenly coated with thebacterial culture.

d. Replace cover on culture dish.e. Throw the used sterile swab in the biohazard waste container.

4. Next, the isolated spice oil will be placed in contact with the inoculated culture.a. Add the required volume of acetone to your oil sample to make 5 to 10%

(wt/vol) solutions of your isolated oil in acetone.b. Insert the tweezers into the alcohol beaker to sterilize.

c. Remove tweezers from beaker and fan back and forth to evaporate thealcohol.

d. Pick up one sterile sample disk using the sterile tweezers and hold it incontact with the solution of your isolated spice. Allow the solvent to dryand then transfer the disk to the culture disk by lifting the lid slightly andplacing it in the center of one of the 4 quadrants.

5. Next, transfer a control disk to the culture dish.a. Transfer the disk to a culture plate by slightly lifting the lid and placing it

in the center of the remaining quadrant without touching the sides of thedish.

b. Wrap the edges of the dish with Parafilm.c. Allow the dish to sit on the bench for about 10 minutes to seat the disks in

the agar.6. Invert a culture plate so that the bottom is facing up.

Place the plate in your locker. The results of the assay can be assessed after 24 hours orlonger.

Figure 2. An agar plate, inoculated withbacteria, with test disks (clockwise fromtop left) of: control, 2 mg of clove oil, 2mg of neutral compounds from clove oil,

and 2 mg of eugenol from clove oil.

Cleaning UpIt is important to properly dispose of the chemical and biological wastes. The aqueousacid and base solutions can be poured down the drain with plenty of running water. Theacetone solutions of spice oil and the neutral components can go in the NHO container.

The dichloromethane solutions used for GC/GC-MS should go in the HO container.Please refer to “Working with Biohazards” described above for bioassay waste disposal.

POSTLAB QUESTION

What criteria would you use to decide whether distillation is an appropriate technique forthe purification of a reaction product and what type of distillation method – simple,fractional vacuum or steam – should be used.

REFERENCES

(1) Introduction to Organic Laboratory Techniques A Microscale Approach, DonaldL. Pavia, Gary M. Lampman, George S. Kriz, Randall G. Engel, SaundersCollege Publishing, pgs. 489, 667.

(2) William H. Miles, and Patricia M. Smiley, J. Chem. Ed. 2002 79, 90

(3) Palleros, D. R. Experimental Organic Chemistry; John Wiley & Sons, Inc.: NewYork, 2000.

(4) Lehman, J. W. Operational Organic Chemistry: A Problem-Solving Approach tothe Laboratory Course, 3rd ed.; Prentice Hall: Saddle River, New Jersey, 1999.

(5) Ntamila, M. S.; Hassanali, A. J. Chem. Ed. 1976, 53, 263