Chapter 9

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

Stereoisomerism
Objectives
• 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
• There is a podcast and a pre-lab assignment for this experiment. Read pages 141-157 of
Padias, and pages 188-214, 1182-1188, and 1257-1261 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.
Safety
• 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:
PT = P A + PB 9-1
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 In equation 9-1, PT is the total vapor pressure and PA and PB 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, Po:
PT = XAPoA + XBPoB 9-2
The mixture will boil when PT 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:
YA = PA/PT = XAPoA/(XAPoA + XBPoB) 9-3
In equation 9-3 YA is the mole fraction of A in the vapor phase in equilibrium with the
liquid. According to equation 8-3 YA > XA if PoA > PoB. 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.

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 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:
PT = PoA + PoH2O 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 PT 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 20-100 torr, so the vapor
pressure of water at the boiling point of the mixture will be around 660-740 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:
YA = PoA/(PoA + PoH2O) 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 set-
up 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.

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 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

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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 sp2 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 CH2Cl2
(DCM) and add this solution to the separatory funnel. Stopper the separatory funnel, invert

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(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 MgSO4 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 10-15 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 Rf 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 Rf values for the six standards. Make
another table of Rf 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 Rf 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.

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 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, 665.
2. Fortineau, A.-D. J. Chem. Educ. 2004, 81, 45.
3. Mannschreck, A.; von Angerer, E. J. Chem. Educ. 2011, 88, 1501.
4. Ciaccio, J. A.; Alam, R.; D’Agrosa, C. D.; Deal, A. E.; Marcelin, D. J. Chem. Educ. 2013,
90, in press. DOI: 10.1021/ed300348k

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