Chapter 9 EXPERIMENT #7: Steam Distillation of Essential Oils: TLC Analysis and Stereoisomerism

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1 Objectives Chapter 9 EXPERIMENT #7: Steam Distillation of Essential Oils: TLC Analysis and Stereoisomerism To learn how volatile oils can be isolated from non-volatile components by steam distillation To perform a steam distillation to isolate an essential oil and to analyze the oil by TLC To identify the sources of stereoisomerism in natural products The Assignment Safety There is a podcast and a pre-lab assignment for this experiment. Read pages of Padias, and pages , , and of Klein. Students work in pairs to perform the steam distillation, and to obtain the IR spectrum. TLC procedures and data will be developed and shared with other groups distilling different products. Students will turn in the notebook pages for this experiment with answers to questions as a summary report. Anise, caraway, cinnamon, catnip, clove, and spearmint oils are commercial products. If you have an allergy to any these products, let your TA know. Isolation of the oils from the distillate requires extraction with dichloromethane (DCM). DCM is an acute inhalation hazard and can lead to skin and eye irritation. Long term exposure has led to cancer in laboratory animals. Wear gloves and goggles, and use DCM only in the hoods. DCM waste must be placed in the solvent waste containers in the hood. Introduction Distillation Distillation is a separation and purification technique that separates components from liquid mixtures based on their vapor pressure. The technique requires that the component(s) being purified go from the liquid to gas phase during purification. To do this the materials undergoing distillation must have appreciable vapor pressures at temperatures below their decomposition points. This requirement can lead to complications in the purification of very high boiling liquids that decompose at temperatures near their atmospheric boiling points. There are techniques to avoid this problem, one of which will be made use of in this experiment. Before we get to that, we need to consider why distillations work in the first place. The pressure and composition of a vapor above an ideal mixture of liquids can be calculated based on Rault s and Dalton s laws. According to Rault s law the vapor pressure above such a mixture is the sum of the vapor pressures of the individual components. For a two component mixture we would have: P T = P A + P B 9-1 Page 1 of 7

2 In equation 9-1, P T is the total vapor pressure and P A and P B are the vapor pressures of the two components. The pressures of the two components are governed by their mole fractions, X, in the liquid, and the equilibrium vapor pressures of the pure components, P o : P T = X A P o A + X B P o B 9-2 The mixture will boil when P T is equal to atmospheric pressure. The composition of each component of the vapor in equilibrium with the liquid mixture is given by Dalton s law: Y A = P A /P T = X A P o A/(X A P o A + X B P o B) 9-3 In equation 9-3 Y A is the mole fraction of A in the vapor phase in equilibrium with the liquid. According to equation 8-3 Y A > X A if P o A > P o B. Most distillation techniques rely on the fact that the substance with the greatest vapor pressure will be enriched in the vapor phase in equilibrium with a boiling liquid phase containing a mixture of substances. Condensation of the vapor phase leads to a sample containing a greater proportion of the more volatile component. A simple distillation is a procedure in which this liquid-vapor equilibrium occurs once. This procedure will ordinarily lead to only partial separation of two volatile components unless one of them has much greater vapor pressure than the other. If one component of a two-component mixture has no measurable volatility this method will lead to a satisfactory separation of both components. Fractional distillation is a technique that sets up a series of liquid-vapor equilibria, typically in a long vertical column packed with inert materials containing a large surface area. The multiple phase equilibria established in such a fractionating column may lead to efficient separation of mixtures of two volatile materials provided that there is a difference in their vapor pressures. The separation is achieved because the vapor component of each individual equilibrium becomes the liquid component for the next equilibrium established in the column. As each equilibrium is established, the vapor phase in equilibrium with the liquid phase becomes successively more enriched in the more volatile component. Figure 9-1. A simple steam distillation set-up. Page 2 of 7

3 Simple or fractional distillations work well as long as the components do not decompose at or near their boiling points. If they do, there are two different techniques that can be used to distill such materials. One of these is vacuum distillation in which the atmospheric pressure above the liquid is reduced by applying a vacuum. As a result, the mixture boils at a lower temperature than its atmospheric boiling point and decomposition is avoided. (Rotary evaporation that you used in Experiment #2, and that you will also use in this experiment, is a type of vacuum distillation. In rotary evaporation the point is to isolate the non-volatile materials by rapidly distilling the volatile solvents away under reduced pressure.) A different technique, steam distillation, can be used if the materials to be purified are immiscible in water. This often occurs during the purification of essential oils from various plant sources. In this case the total pressure above the mixture of water and the immiscible liquid is given by equation 9-4: P T = P o A + P o H2O 9-4 Mole fractions of the components do not appear in the equation because water and the immiscible liquid are present in two different liquid phases. When P T equals atmospheric pressure, distillation occurs at a temperature < 100 C because the total pressure of the mixture depends on the vapor pressure of both water and the immiscible liquid. Typically the vapor pressures of essential oil components near 100 C are in the range of torr, so the vapor pressure of water at the boiling point of the mixture will be around torr, below its vapor pressure of 760 torr at 100 C. Typically the boiling point of the mixture will be 1-4 C below the boiling point of pure water. The mole fraction of the natural product in the vapor will be given by equation 9-5: Y A = P o A/(P o A + P o H2O) 9-5 Typically the mole fraction of the immiscible liquid in the vapor will be around 0.03 to 0.15, so a substantial amount of the distillate will be the desired material. Since the two liquids are immiscible, they can be separated from each other by extraction. A simple steam distillation setup that you will use in this experiment is illustrated in Figure 9-1. Essential oils Chart 9-1. Major components of essential oils from six different sources. Page 3 of 7

4 Essential oils are water insoluble volatile natural product mixtures obtained from plant sources that are used in perfumery, as food additives, as insecticides, and for medicinal purposes. The traditional method for obtaining these oils was steam distillation which is still used to obtain many commercially available essential oils. Many essential oils are complex mixtures of compounds in which no single material predominates. Other essential oils may be composed of 70-90% of a single compound with smaller amounts of other materials. In this experiment we will concentrate on the latter group of essential oils. Chart 9-1 shows the major constituent of several essential oils that will be obtained by steam distillation in this experiment. You will notice similarities in the structures of many of these compounds. The first three compounds, anethole, cinnamaldehyde, and eugenol, are characterized by an aromatic ring with an unbranched 3-carbon side chain. They belong to a class of naturally occurring compounds called phenylpropenes, all of which are derived by metabolism of the amino acid phenylalanine (Chart 9-2). The other three compounds, nepetalactone, (+)-carvone, and (-)-carvone, are examples of natural products called terpenes that are ultimately derived from the five carbon isoprene unit by metabolic pathways involving isopentenyl pyrophosphate and dimethylallyl pyrophosphate (Chart 9-2). Terpenes such as nepetalactone and the two carvone isomers that contain 10 carbons are biosynthesized from two isoprene units, and are referred to as monoterpenes. Sesquiterpenes are composed of three isoprene units and have 15 carbons. Diterpenes contain 20 carbons from 4 isoprene units, sesterterpenes (a rare group) contain 25 carbons, triterpenes contain 30 carbons, sesquarterpenes (mostly microbial in origin) contain 35 carbons, and tetraterpenes contain 40 carbons from 8 isoprene units. You will become familiar with the structure of one tetraterpene, β-carotene, and the diterpenes retinol, retinal, and retinoic acid in Experiment #9 this semester. Chart 9-2. Biological sources of phenylpropenes and terpenes Stereoisomerism in natural products such as these can arise from the presence of chirality centers (also known as chiral centers or asymmetric centers), a tetrahedral atom bearing four different substituents, or stereocenters (also known as stereogenic centers), an atom at which the exchange of two substituents will generate a stereoisomer. There are examples of both of these in the compounds shown in Chart 9-1. Chirality centers are a subset of the more general concept of stereocenters. A single chirality center in a molecule gives rise to enantiomers, stereoiomers that are mirror images of each other: the two isomeric carvones are examples of enantiomers generated by a single chirality center. Multiple chirality centers or stereocenters in a molecule Page 4 of 7

5 gives rise to diastereomers, stereoisomers that are not mirror images of each other. There is a naturally occurring diastereomer of nepetalactone, also present in catnip oil, in which the stereochemistry of the chirality center adjacent to the carboxyl carbon of the lactone is inverted. Anethole has stereocenters at the two sp 2 carbons of the prop-2-enyl side chain. Exchange of the substituents at either of those carbons generates the Z-isomer, the diastereomer of the E-isomer that is the predominant isomer in anise oil. The fact that humans can distinguish the odors of (+)- and (-)-carvone is proof that the olfactory receptors are chiral in nature. In this experiment students will isolate the essential oils from six different plants sources by steam distillation. Each pair of students will perform only one steam distillation. Following the steam distillation, students will obtain an IR spectrum of the oil. Six pairs of students will work together to develop a common TLC solvent mixture that can be utilized for the analysis of all the essential oils so that direct TLC comparisons can be made among the oils. In the summary report questions will be answered about the composition of each oil from the TLC results, and the IR spectra. Questions about stereoisomerism in the major constituents of each oil, and in related compounds, will also be answered. The Experiment Steam Distillation TAs will assign each pair of students one of the crude plant materials with which to perform the steam distillation. Weigh and record in your notebook 15.0 ± 0.1 g of your assigned source material. Transfer the material into a 500 ml round bottom flask that will serve as the distillation flask. Set up the distillation apparatus as shown in Figure 9-1. Use a 250 ml round bottom flask as the receiving flask. Lightly grease joints before setting up the apparatus. Remember to put a stir bar into the distillation flask. Note the position of the thermometer in the distillation head, and make sure that hoses are properly attached to the condenser. The inlet hose must be connected to a water source, and the outlet hose must lead to a sink. Do not turn the condenser water on until the apparatus is completely set up and the distillation is ready to start. Clamp the apparatus securely where indicated in Figure 9-1 with the distillation flask positioned about 3-5 mm above the surface of the heater stirrer. Remove the stopper from the Claisen adapter, and add 300 ml of water to the distillation flask with a funnel. (For the distillation of cinnamon oil only add 4 ml of 6 M HCl to the distillation flask in addition to the 300 ml of water. This helps to reduce foaming during the distillation.) Turn on the condenser water to a slow flow rate, turn the heat on the heater stirrer to about mid-range and turn the magnetic stirrer motor to a low setting so that slow stirring occurs in the distillation flask. Once boiling of the mixture starts and the distillate starts to condense on the thermometer, adjust the heater control to obtain a distillation rate of about 2-3 ml/min. Distill until you have collected about 100 ml of distillate, or until the distillate appears to run clear without any evidence of a second phase. Stop the distillation by turning the hot plate off, remove the receiving flask after distillate has stopped collecting and cool the flask in an ice-water bath. Disassemble and clean the rest of the apparatus after it has cooled. The remaining water in the distillation flask should be poured into the solvent waste container in the hood, and the solids remaining in the flask should be placed into the solid waste container in the hood. While the distillation apparatus is cooling transfer your distillate with a funnel into the 125 ml separatory funnel situated in a ring clamp. Rinse the receiver flask with 10 ml of CH 2 Cl 2 (DCM) and add this solution to the separatory funnel. Stopper the separatory funnel, invert Page 5 of 7

6 (Figure 4-2), vent the funnel momentarily, and shake gently for about 2 min, venting periodically to avoid pressure build up. Return the separatory funnel to the ring clamp and allow the layers to separate. Drain the DCM layer (which one is it?) into a clean, dry 125 ml Erlenmeyer flask, and extract the aqueous solution with a second 10 ml of DCM. Combine the second DCM extract with the first one, and add about one spatula tip of anhydrous MgSO 4 to the Erlenmeyer flask. Swirl gently, and make sure that some of the drying agent remains free flowing. If not, add more drying agent until some of it remains free flowing after swirling. Allow the DCM extract to stand for about min and then transfer it by gravity filtration into a tarred, clean, dry 50 ml round bottom flask. Wash the drying agent in the Erlenmeyer flask with 5 ml of DCM, and add this solvent to the round bottom flask through the gravity filtration funnel. Evaporate the dried DCM solution on the rotary evaporator, and obtain the mass of the flask containing the essential oil. Determine the yield of the oil, and obtain an IR spectrum of the oil. Remember that each pair of students will need four copies of the IR spectrum, two copies to be permanently attached to both notebooks and two copies for submission with the summary report. Each pair of students will work together with five other pairs who distilled the five other oils to develop a common TLC procedure for the analysis of the oils. Use the silica gel plates with fluorescent indicator that you first used in Experiment #4. Find a mixture of hexanes/etoac that provides reasonable R f values (ca. 0.3 to 0.8) for all major components of the oils with good separation for the standard samples of the six compounds that are listed in Chart 9-1. The standard samples will be provided to you in lab as solutions in EtOAc. After an agreed upon solvent mixture has been found, perform TLC analyses on the six oils, along with the six standards. Share data within the group, and make a table of R f values for the six standards. Make another table of R f values for all major components of the essential oils (some oils will have more than one easily observed component upon visualization). The Report Do all of the following in your notebook as the summary report. 1. Calculate the % recovery of your essential oil based on the dry weight of the sample of plant material you used in the steam distillation. 2. Examine the IR spectrum of your oil. Make a table of the frequencies of all the significant bands observed in the spectrum. Assign these bands to a functional group/bond type in a second column of the table. In a third column in the table indicate whether each band is consistent with the main component of your oil listed in Chart 9-1. What additional functional groups of other compounds, if any, appear to be in your oil? 3. Use the combined TLC data to identify the spot on the TLC of each oil that corresponds to the major component of that oil. Are there any ambiguities in your assignments? What is the basis of any ambiguity? Is it possible to identify any of the other components of the oils based on the R f values for the six standards? If that is the case do so. 4. Draw the structures of the six materials in Chart 9-1 in your notebook. Draw a square around any chirality center in all of the molecules, and identify the absolute stereochemistry as either R or S. Draw a circle around any stereocenters that are not also chirality centers. Now draw all the stereoisomers of the six structures in Chart 9-1 and identify them as diastereomers or enantiomers of the original structure. Page 6 of 7

7 5. Several other natural products are drawn below. Redraw these in your notebook and perform the same stereochemical analyses on these compounds that you were asked to do in Question #4. References 1. O Shea, S. K.; Von Riesen, D. D.; Rossi, L. L. J. Chem. Educ. 2012, 89, Fortineau, A.-D. J. Chem. Educ. 2004, 81, Mannschreck, A.; von Angerer, E. J. Chem. Educ. 2011, 88, Ciaccio, J. A.; Alam, R.; D Agrosa, C. D.; Deal, A. E.; Marcelin, D. J. Chem. Educ. 2013, 90, in press. DOI: /ed300348k Page 7 of 7