Steam Distillation of an Essential Oil

Your preparations should be routine by now. Prepare your notebook by:

• drawing structural formulas of the compounds that might be found in clove oil, write each compound’s name under its formula.
• constructing a table of physical properties (MW, mp, bp, d) for the compounds that you might handle in this experiment (compounds present in your essential oil, diethyl ether, anhydrous sodium sulfate, ethyl acetate, hexanes, silica gel, chloroform-d, acetone)
• listing any unusual hazards that a lab worker should be aware of

Hazard. Silica gel is a fine powder that, inhaled over time, causes a disease called “white lung” or silicosis. All operations with this powder should be performed in a fume hood. Also, the powder should be disposed of in a special container (located in same hood where it is dispensed).

• listing disposal information for all compounds

Online videos. Please review at least one online video on thin-layer chromatography (TLC) and another on rotary evaporation (rotovap). In addition to Moodle, here are some video options:

• Thin-layer Chromatography (TLC) – The Interactive Lab Primer
• Thin-layer Chromatography (TLC) – MIT
• Rotary Evaporation (“rotovap”) – The Interactive Lab Primer
• Rotary Evaporation (“rotovap”) – MIT

Grinding. Obtain about 25 g of cloves. Grind/crush them gently with a mortar-and-pestle to break down the material so that water can penetrate the material more easily. It is advised to research what part of the material the oil is mainly found in and don’t worry if you can’t easily grind up or cut other parts of the plant that don’t contain oil. Grinding to a fine powder is not recommended because it seems to make the boiling liquid foam up. Foams make distillations more difficult, partly because they slow down liquid-vapor equilibration, and partly because they require a reduction in heating in order to prevent the foam from entering the Claisen head, condenser, etc.

Steam distillation. Transfer the material and any liquids produced to a 250 mL round bottom flask. This will be your boiling flask for the simple distillation. Add roughly 100 mL of deionized water and a stir bar to the boiling flask and clamp the flask to the monkey bars above a ceramic heater, stirring motor, and lab jack. Set up the rest of your simple distillation apparatus. Collect your distillate in a 100 mL graduated cylinder. During the distillation, periodically transfer your distillate into an Erlenmeyer flask and cover until 100 mL has been collected. Try not to let dry plant material ‘bake’ anywhere in the flask. If the material becomes dry and/or the water level falls too low, lower the heater, use a DI water bottle to rinse plant material off the sides of the flask and to cover the plant material, and then resume heating. To avoid excessive foaming, insulate your apparatus and monitor it throughout the distillation so that you can adjust the heat. When your distillation is finished, shut off your heater, and allow the apparatus to cool to room temperature.

Liquid-liquid extraction. Transfer your distillate (approx. 100 mL) into your 250 mL separatory funnel. Extract the aqueous layer three times with diethyl ether (approx. 20 mL per extraction). Dry your combined diethyl ether layers over anhydrous sodium sulfate. To dry the solution, slowly add the Na₂SO₄ (1 g at a time) until you reach the stage where no visible clumping is occurring. Leave the solution to sit covered for 15 minutes. Carefully decant your dried ether solution into a dry, pre-weighed 250 mL round bottom flask. Save approximately 0.5 mL of your final dried ether solution in a capped vial for TLC analysis during Weeks 2 and 3. If you have time you can attempt the TLC analysis during Week 1. Check in with a TA or instructor before you begin.

Evaporation. Use the rotovap to remove the diethyl ether solvent from your sample to give the crude essential oil mixture. Once the ether has been evaporated, weigh your rbf then stopper it, wrap the joint in plastic, and store it in your cabinet until Week 2.

The goal of this TLC analysis will be to learn how many compounds are present in your sample and to practice good TLC technique. TLC analysis requires very little material (microliters) and even then the samples must be diluted in a volatile solvent. You should never apply pure compounds to a TLC plate. The molecules in the sample will spread across the plate until they can find sites on the silica gel to bind to creating large spots that are hard to analyze. Smaller sample spots are better.

To analyze your ether solution by TLC, start by selecting a TLC plate and drawing a baseline approximately 1 cm from the bottom of the plate in pencil. Do not score the silica. Next, make three equally spaced hash marks on the baseline.  On the first and second marks, spot a small drop of the ether solution from the liquid-liquid extraction onto the baseline using a capillary tube. The second mark will be the “co-spot.” Next, make a standard solution of your commercial essential oil by dissolving one drop in 1 mL of ethyl acetate. Then spot the TLC standard solution of your oil on the baseline at both the second and third marks. Standard solutions are fairly concentrated so apply just one small drop from the capillary tube for each of them.

Using forceps, carefully place the plate in a 200 mL tall form beaker with about 0.3 cm of developing solvent (80:20 hexanes:ethyl acetate). Cover the beaker with a watch glass and allow the solvent to rise to within 1 cm of the top of the plate. Using forceps again, remove the plate, carefully mark the top of the solvent front with a pencil, and allow the plate to air dry. View the plate using a UV light and record any spots and their locations in your lab notebook by drawing the plate and measuring the distances any spots traveled from the baseline with a ruler. Your DRAWING is your most reliable data record (see Padias Figure 3-29). Be as precise as you can be when drawing your plate + spots. Do not just make a rough sketch.

Next, we will use a chemical visualization reagent. This is a mixture of 2.5% 4-anisaldehyde, 1% acetic acid, and 3.5% sulfuric acid in 95% ethanol) and is highly corrosive and should only be used on a suitable surface and in a fume hood. Dip the plate in the visualization reagent, then heat the plate with a heat gun, and record the positions and shapes of any colored spots that appear.

Once you are finished with the first TLC plate, determine if you need to adjust the polarity of the solvent mixture. Record any observations.

TLC troubleshooting. Did the two visualization methods provide the same or different information about your oil? Do they provide the same or different information about the “pure” samples you are comparing your oil to? If they provide the same information, it may be unnecessary to use both visualization methods during the dry column flash chromatography procedure. Otherwise, both are required.

Are your spots small and faint, or have they formed large (and possibly overlapping) streaks on the plate? If the latter, perhaps your original spots were too large or perhaps your original samples were too concentrated. If the former, perhaps you submerged the original spot in the elution solvent when you inserted your plate into the developing chamber? Or perhaps your samples were too dilute.

Your TLC analysis has probably revealed that the oil contains at least two compounds and possibly more. Column chromatography is a useful technique for separating these compounds in sufficient quantities so that they can be characterized by NMR and/or IR spectroscopy.

A full chromatographic separation consists of several distinct steps:

• preparing a column
• applying a sample to the top of the column
• eluting the components of the oil from the bottom of the column; this is accomplished by passing batches of solvent (“fractions”) through the column
• analyzing the collected fractions by TLC to determine whether they contain any oil compounds
• combining fractions that contain a single compound and evaporating the solvent from the combined fractions
• weighing the recovered compound and characterizing it spectroscopically

Our goal for this experiment is for you to elute the major component of the oil you isolated and to analyze it. The steps are outlined in greater detail below.

Preparing your column. A “dry flash column” consists of a sintered glass funnel tightly packed with TLC-grade silica gel. The silica gel fills most, but not all, of the funnel. There needs to be roughly 1 cm of “head space” at the top of column so that solvent can be applied to the silica gel without overflowing the walls of the funnel.

Loosely fill a sintered glass funnel to the lip with dry TLC-grade silica. CAUTION – silica powder is harmful if inhaled. Work carefully with this material and use it only in a fume hood.

Place your funnel on top of your filter flask (see below; click on image for larger image). The funnel should be supported by one or more neoprene conical adaptors to create a gas-tight connection between the funnel and the flask, and also to adjust the height of the funnel’s spout inside the test tubes that will be used to collect solvent fractions.

Tap the funnel gently on all sides to settle the powder and to remove any voids. A heavy rubber hose or cork ring makes a nice ‘tapping’ tool. Hook up a hose from the vacuum pump, apply suction, and continue tapping the funnel gently on all sides to remove voids. Next, while still applying suction, press down on the top of the silica bed, carefully, but firmly, with a flat stopper, rubber cork, or small beaker. Carefully work around the perimeter first and then in towards the center to produce a level, well-compacted bed. The goal is a reasonably flat, compacted surface with about 1-0.5 cm of head space for the addition of the mixture and solvent fractions.

Testing your column. Before you apply your sample to the column, it is necessary to make sure that solvent will flow through the column without causing it to collapse.

Place a small piece of filter paper (4 cm diameter) on top of the column. This will protect the surface from the solvent that you are about to pour on it. The impact of falling solvent tends to erode a small crater at the center of the column. Filter paper reduces this problem by directing the solvent flow to the edges of the paper.

Apply suction to the column. Then pour hexanes on the column at a sufficient rate to keep the entire surface of the filter paper covered with solvent. While you are pouring hexanes on the filter paper, notice how the solvent flows through the column. Ideally, the solvent “front” (the lower edge of the advancing solvent) will be horizontal indicating simultaneous and equal flow of solvent through all parts of the column. Once the column is entirely wet, stop adding solvent, and maintain suction until the flow is reduced to a few drops. If you have trouble establishing a good flow of hexanes, or monitoring the solvent front, stop adding hexane and apply suction until the column runs dry. Then try again.

Finally, examine the “dry” column for channels, voids, holes, etc. If you find any defects like these, discard the silica gel in the special waste container provided, build a new column with fresh silica gel, and repeat the test. If your column passes the test, that is, if it is well-packed and passes solvent without problems, move on to the next step.

Preparing your sample. Dissolve ~400 mg of your product mixture (record the actual weight in your notebook) from Week 1 in 3-4 ml of hexanes. If the mixture fails to form a homogeneous solution, add ethyl acetate dropwise until it does. An apparatus of this size has a limited separating capacity. 400 mg is the upper limit on samples that are difficult to moderately difficult to separate. Slightly larger samples, 500-600 mg, can be processed if they are easily separated. This particular procedure gives a 5-10% w/v solution of oil in hexanes. Harwood’s original instructions stated that the sample should be applied as a “25% w/v solution in the initial solvent”. Although your initial solvent is hexanes, it is not always possible to make up a 25% w/v solution in hexanes because many compounds are only somewhat soluble in hexanes. Including a small amount of ethyl acetate does not adversely affect the separation.

Applying your sample to the column. Remove the filter paper from your column. Using a pipet, apply the oil-hexanes solution evenly across the top of your column dropwise, then apply suction. When the column has been sucked “dry”, place the filter paper back on top, and move on to the next step.

Elute the oil compounds from the column. The general procedure for eluting involves these steps:

• Place a clean test tube in the filter flask
• Replace the funnel so that the drip tube extends partway into the test tube
• Apply strong suction (it may be necessary to adjust the funnel so that it makes a tight seal with the filter flask)
• Pour solvent evenly on top of the column until the test tube is partly full (about 25 mL; do not overfill)
• Carefully pry the funnel (or the rubber hose) from the flask to release the vacuum
• Remove and store the “filled” test tube

This procedure is repeated as many times as is necessary to elute all of the oil compounds of interest from the column. Because the compounds are invisible as they move through the column, it will be necessary to analyze each fraction using TLC to detect the compounds. This means there are two experimental parameters that you can play with: the number of fractions that you collect and the polarity of the solvent used in each fraction. As a general rule, collect 22 fractions before you stop to analyze any of them. If more fractions are needed, collect at least 4-8 more fractions before stopping to analyze the next batch. As a second general rule, use pure hexanes (H) for your initial fractions, and then slowly increase the polarity of subsequent fractions by adding increasing amounts of ethyl acetate (EA) to the hexanes. A procedure for creating fractions of appropriate polarity is described next.

Slowly increase the polarity of your fractions. For this experiment, make fractions 1-3 pure hexanes. After preparing the apparatus as described above, apply suction, and pour pure hexanes through the column. Remove the suction when 20-25 mL of liquid has been collected and replace the test tube with a clean one. Repeat this procedure one more time, then continue with the solvent mixtures described below and record the composition of each solvent mixture in your lab notebook:

Fractions 3-6:

• Fraction 3: Pour 100 mL H into a 100 mL graduate cylinder. Pour solvent from the cylinder into the funnel until ~25 mL has been collected.
• Fraction 4: Add 1 pipet load EA. 1 pipet load to the remaining liquid in the graduated cylinder. 1 pipet load is roughly 1 mL. Mix this liquid thoroughly. You can mix the liquid by pouring it back and forth between the graduated cylinder and another container, say, a 200 mL Erlenmeyer or beaker. Pour solvent directly from the cylinder into the funnel until another 25 mL has been collected.
• Fraction 5: Repeat the procedure for fraction 4 except use ~0.5 pipet load EA.
• Fraction 6: Repeat the procedure for fraction 5.

Fractions 7-10: The instructions are identical to those for fractions 3-6 except that you begin by pouring 95 mL H and 5 mL EA into a 100 mL graduated cylinder.

Fractions 11-14: The instructions are identical to those for fractions 3-6 except that you begin by pouring 90 mL H and 10 mL EA into a 100 mL graduated cylinder.

Fractions 15-18: The instructions are identical to those for fractions 3-6 except that you begin by pouring 85 mL H and 15 mL EA into a 100 mL graduated cylinder.

Fractions 19-22: The instructions are identical to those for fractions 3-6 except:

• that you begin by pouring 80 mL H and 20 mL EA into a 100 mL graduated cylinder
• Fraction 20: add 2 pipet loads EA to the graduated cylinder; Fractions 21-22: add 1 pipet load EA each time to the graduated cylinder.

Fractions 23-26: The instructions are identical to those for fractions 3-6 except:

• that you begin by pouring 70 mL H and 30 mL EA into a 100 mL graduated cylinder
• Fraction 24: add 3 pipet loads EA to the graduated cylinder; Fractions 25-26: add 2 pipet loads EA each time to the graduated cylinder.

Fractions 27 and beyond: Use a 50:50 H:EA mixture for all subsequent fractions.

Analyze the fractions by TLC. Once you collect 22 (or how ever many) fractions, test them by applying a small sample of each to a single fluorescent TLC plate (label your spot locations!) The fractions that contain your compound are very dilute. Therefore, apply all of the liquid in your capillary tube to the TLC plate. It is necessary to keep the spot very small, so do the following: quickly touch the capillary tube to the plate, blow on the plate to evaporate the solvent, then touch the capillary to the same spot to add more sample. Always let the solvent evaporate before applying more sample.

Do not elute the plate. Instead, examine the plate under a UV light to see which fractions contain a UV-absorbing compound. Also test your samples using the dip reagent. Record these data in your notebook.

If any of your fractions test positive (UV or dip reagent), analyze these samples by standard TLC, i.e., by spotting them near one end of a TLC plate, eluting the plate with an appropriate solvent, and detecting the results using UV and dip reagent. Test only the fractions that positively contain a compound. Remember that these “positive” fractions are very dilute so you will need to apply all of the liquid in the capillary tube to the TLC plate. You can also spot the eugenol and/or isoeugenol TLC standard solutions onto your plate as well. Elute the plate in the standard way using whatever solvent mixture and visualization method had resolved your oil compounds previously. Record the results in your notebook.

Isolate and characterize the major compound. Combine the fractions for the major component of your essential oil into a pre-weighed 250 mL r.b.f. Choose the fractions based on the size and intensity of the spots your observed during your TLC analysis. Do not fill the flask more than halfway full. Concentrate your sample using the rotovap. Weigh your final product, then prepare an FT-NMR sample by dissolving 10-20 mg of your oil in 0.5 mL of chloroform-d. Collect a neat FT-IR of your sample.

Once your are done, the remainder of the purified oil as well as the crude oil from Week 1 can be transferred into a vial and taken home! If you don’t want your essential oil, dispose of it in organic waste.