In the Laboratory
W
An Engaging Illustration of the Physical Differences
among Menthol Stereoisomers
Edward M. Treadwell* and T. Howard Black
Department of Chemistry, Eastern Illinois University, Charleston, IL 61920-3099; *cfemt@eiu.edu
One of the important concepts introduced early in organic chemistry courses is stereochemistry, which is often difficult for students to grasp. Students struggle with the
mechanics of assigning absolute stereochemistry and determining the relationship between two stereoisomers, as well
as the broader implications of stereoisomerism in terms of
chemical and physical behavior. Demonstrations and laboratory exercises can be of great assistance in reinforcing these
ideas. Classic classroom demonstrations of the difference in
behavior of enantiomers include overhead polarimetric demonstrations using either carvone or limonene, and the “sniffing” of carvone enantiomers (1). Most laboratory experiments
incorporating stereochemical concepts involve completion of
a reaction and comparison of the differing physical properties of either the enantiomeric or the diastereomeric products. Some of these reaction-based experiments involve optical
resolutions of a racemic starting material where the separated
enantiomers are compared to each other (2). Alternatively,
stereospecific reactions to produce a single diastereomeric
product, or stereoselective reactions to give a mixture of major and minor diastereomeric products are employed (3). In
these stereospecific and stereoselective experiments, the difference in physical properties of diastereomers is clearly evident given the melting point and spectral data.
We wished to devise an experiment illustrating stereochemical principles that did not require a reaction, principally to allow implementation concurrent with discussion of
stereoisomers in the lecture class without having to introduce
reactions that had yet to be covered. Previously, we had been
using an experiment employing cis- and trans-1,2-cyclohexanediol, where the diastereomers are differentiated by TLC
analysis (4); unfortunately, the difference in Rf values is very
small, and the chemicals are quite expensive.1 The expense
would be exacerbated significantly if the experiment was expanded to include the use of enantiomers, as the nonracemic
trans-1,2-cyclohexanediol stereoisomers cost over $150g in
the most recent Aldrich catalog.
We sought a compound where several stereoisomers were
commercially available, relatively inexpensive, preferably nontoxic, and (for the diastereomers) possessing significantly different physical properties. An attractive candidate was found
in a series of menthol stereoisomers (5). (+)-Menthol, (−)menthol, (+)-neomenthol, and (+)-isomenthol are all commercially available, with the most expensive of the four isomers
being (+)-neomenthol at five grams for $55, or $11 per gram.
This set of four compounds (Figure 1) is composed of a pair
of menthol enantiomers, along with two different diastereomers [(+)-neomenthol and (+)-isomenthol]. Menthol is also
attractive from a pedagogical standpoint since it is a compound
with numerous everyday uses, including as an ingredient in
cough drops, lip balms, cigarettes, perfumes, and liqueurs (6).
We then devised an experiment that was intended to have
the students learn: (i) that diastereomers have different physi1046
Journal of Chemical Education
•
CH3
CH3
OH
CH3
CH3
OH
CH3
CH3
(ⴚ)-menthol
mp = 43 – 45 °C
(ⴙ)-menthol
mp = 43– 45 °C
CH3
CH3
OH
CH3
CH3
(ⴙ)-isomenthol
mp = 77–83 °C
OH
CH3
CH3
(ⴙ)-neomenthol
mp = ⴚ22 °C
Figure 1. Structure of commercially-available menthol (2-isopropyl5-methylcyclohexanol) stereoisomers.
cal properties using TLC and melting point analysis; (ii) that
enantiomers have the same physical properties in achiral environments (TLC analysis) but different properties in chiral
environments (polarimetry and mixed melting points); (iii)
how to assign absolute stereochemistry; and (iv) how to relate the stereoisomeric relationships to differences in absolute stereochemistry.
Experimental Overview
An easily observed difference between the diastereomers
is their melting points. In the laboratory, the melting points
are sufficiently distinct to allow clear identification, as each
menthol enantiomer melts at 43–45 ⬚C while (+)-isomenthol
melts at 77–83 ⬚C and (+)-neomenthol is a liquid at room
temperature.2
Thin-layer chromatographic analysis was also performed
on all the menthol isomers, using a vanillin solution for visualization. First, the students preformed a TLC analysis on
the samples of (+)-menthol and (−)-menthol to find that the
enantiomers traveled an identical distance, exhibiting an identical Rf value of 0.46.3 Then, the students preformed a TLC
analysis of (+)-menthol, (+)-neomenthol, and (+)-isomenthol
to determine that the diastereomers displayed different Rf
values (0.59 for neomenthol and 0.51 for isomenthol) not
only from the (+)-menthol, but also from each other.
To demonstrate the differences between the enantiomers,
the optical rotations, [α]25D, of (+)- and (−)-menthol in 95%
ethanol were obtained at lower concentrations than previ-
Vol. 82 No. 7 July 2005
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Supplemental Material
Lab Documentation for JCE Online
Notes for Students
Properties of Stereoisomers of Menthol
(-)-Menthol is a naturally occurring compound found in peppermint leaves and other mint plants,
and widely used in cough drops, lip balms, nasal inhalers, liquers, perfumery, and cigarettes. Since
menthol has three stereocenters, there are eight total stereoisomers of this compound. In this experiment
you will observe the differences in the physical behavior of four of the possible stereoisomers : (-)menthol, (+)-menthol, (+)-isomenthol, and (+)-neomenthol. These four compounds include a pair of
enantiomers - (+)- and (-)-menthol - and two diastereomers - (+)-isomenthol and (+)-neomenthol.
CH3
CH3
OH
CH3
CH3
(-)-menthol
CH3
OH
CH3
CH3
(+)-menthol
CH3
OH
CH3
CH3
(+)-isomenthol
OH
CH3
CH3
(+)-neomenthol
Thin-Layer Chromatography (TLC):
Obtain a 10 x 2.5 cm TLC plate (be careful not to touch the silica gel), and, using a pencil, lightly
trace a straight line across the bottom of the plate approximately 1 cm from the bottom. Gently make
three tick marks evenly spaced perpendicular to your first line. Using a TLC spotter, apply a small
sample of the prepared solution of (+)-neomenthol to the first spot on the TLC plate. Using different
spotters, repeat the process for the second spot with (+)-menthol and the third spot with (+)-isomenthol.
Obtain 10 mL of a 4:1 hexanes:ethyl acetate developing solvent, and pour about 7 mL of solvent into
a 250 mL beaker. Rest a piece of cut or folded filter paper against the side of the beaker and gently swirl
the solution to wet the filter paper. Gently place the TLC plate in the beaker (such that the end coated in
silica gel is in the solution), and cover the beaker with a watchglass. The solvent level must be below the
line you drew on the TLC plate. Periodically check the TLC plate, and, when the solvent has reached
near the top of the silica gel on the plate, remove the TLC plate from the beaker, mark the solvent front
with a pencil, and allow it to air-dry with the silica gel side up. Do not allow the solvent front to reach
the top of the absorbent!
Obtain a second TLC plate (be careful not to touch the silica gel), lightly trace a straight line
across the bottom, and gently make two tick marks. Using TLC spotters, apply a small sample of the
prepared solution of (+)-menthol to the first spot, and (-)-menthol to the second spot, and develop the
plate as before.
Take both TLC plates over to the hood, and using the tongs briefly dip each plate into the vanillin
visualization solution. Touch the corner of the TLC plates to a paper towel to allow the excess solution to
drain off, and then heat the TLC plates over a warm hotplate for a few minutes until a blue spot for each
menthol sample is observed. Allow the TLC plates to cool, and measure the distance of the solvent front
and the distance for each menthol spot.
Discard the developing solvent in the Organic Waste container.
Melting points:
First, take and then record the melting point of solid (+)-isomenthol and (+)-menthol. Next, briefly
rub your hands together for a few minutes, and then put on a pair of latex gloves. Have a partner place a
small amount of (-)-menthol on the palm of your left hand, and (+)-menthol an inch or two away. (The
amount of each material should be no bigger than a dime.) Observe what happens (if anything). Then,
using a spatula, push the two solids together and mix them thoroughly. Observe what happens (if
anything). Discard any remaining menthol isomers in the Organic Waste container.
You were not asked to find the melting point of the neomenthol – why is this?
Optical rotation:
Carefully weigh out 30 milligrams of (+) or (-) menthol into a 25 mL Erlenmeyer flask, and using a
volumetric pipet, add 10 mL of 95% ethanol. Remember that both the exact mass of solid used as well as
the exact volume of solvent used will dramatically affect your specific rotation. Gently swirl the flask
for a few minutes to completely dissolve the solid, and then place a cork over your Erlenmeyer flask and
take it to the instrument room to obtain the rotation of your solution. Be sure to write your rotation on the
chalkboard after returning to lab for the rest of the class to use. Pour your solution into the Organic
Waste container.
In addition to describing your results and the rotations in your laboratory report, assign the
absolute stereochemistry at each chiral center for each menthol diastereomer from the structures drawn
above. Be sure to also calculate the Rf’s for each menthol isomer. Finally, include in your discussion
how the physical properties (Rf and mp) are related among the four different stereoisomers.
You may find chapter 9 in your textbook (McMurry’s Organic Chemistry, 5th edition) to be a
helpful resource in writing your report.
Safety notes:
For complete safety information, the MSDS for each chemical should be consulted.
(-)-Menthol (CAS 2216-51-5) – no significant hazards
(+)-Menthol (CAS 15356-60-2) – no significant hazards
(+)-Isomenthol (CAS 23283-97-8) – no significant hazards
(+)-Neomenthol (CAS 2216-52-6) – no significant hazards
Hexanes (CAS 73513-42-5) – flammable, irritant, exposure can affect the central nervous system.
Ethyl acetate (CAS 141-78-6) – flammable, irritant.
Vanillin Developing Solution
Vanillin (CAS 121-33-5) – no significant hazards.
Sulfuric Acid (CAS 7664-93-9) – corrosive, poison.
Ethanol (CAS 64-17-5) – flammable, irritant.
Supplemental Material
Lab Documentation for JCE Online
Notes for Instructor
This laboratory requires only a minimal amount of preparation, and the amounts listed below
were collectively used for two sections of approximately 20 students each. All sections of the laboratory
can be completed in a 2.5 hour lab period. The optical rotation section can be omitted without impacting
the rest of the experiment if a polarimeter is unavailable or cannot be employed due to time constraints.
At the conclusion of the lab, the rotations are copied down by the instructor and the tabulated results are
handed to the students to include in their report.
The student handout is designed to accompany both discussion of stereochemistry in the
accompanying lecture course, as well as a prelab lecture given immediately before the start of the
experiment. The prelab lecture typically dicusses the following topics: (1) what makes a chiral center,
(2) the definition of enantiomers and diastereomers, (3) the difference in physical properties in a set of
enantiomers and in a set of diastereomers, (4) polarimetry, and (5) TLC analysis.
A picture of a developed TLC plate for the chromatography part of the experiment has been
included as an example of a typical separation. Additionally, a photograph of the mixed-melting point
demonstration is provided.
Chemical Preparation
The TLC solutions (2.5%, weight to volume) can be made up in advance (our stockroom
technician prepared a 10 mL sample of each menthol isomer using reagent-grade acetone) and stored in
glass 2 mL vials for extended periods of time, though the neomenthol sample should be stored in a
refrigerator to prevent decomposition. The melting point samples of the (+)- and (-)-menthol must be
finely ground in a mortar and pestle in order for clear results to be obtained (though the mortar and pestle
must be cleaned between uses!). These samples were placed in plastic 0.5 dram vials for student use.
Approximately 500 mL of the 4:1 hexanes:ethyl acetate solution was prepared in a hood, and
placed into four separate bottles. The vanillin staining solution consists of 5% vanillin and 5%
concentrated sulfuric acid in ethanol [v/v],1 and should be prepared in a hood. We typically prepared 240
mL of this reagent and divided it between three 21 cm by 4 cm (inner diameter) jars. It is important that
the jars are full enough so that the whole TLC plate can be immersed in the solution.
Required Materials for Students
- TLC solutions (see above)
- Analtech UniplateTM Silica Gel GF 250 micron TLC plates (2 per student, with a supply of
extras in case of student error)
- Pencils
- TLC spotters (we typical draw our spotters from open-ended capillary tubes)
- TLC developing solution ( 4:1 hexanes:ethyl acetate)
- Forceps to dip plates into visualization solution
- Plastic 6 inch rulers with mm gradations for students to accurately measure
distances with
- 3 or 4 hot plates (in the hood) to visualize the TLC plates
- melting point samples (see above)
- a vial of the neomenthol, or the reagent bottle, for illustrative purposes
- latex gloves
- 95% ethanol (500 mL) in several bottles
- five 10 mL volumetric pipets
- waste bottle for TLC and rotation solutions
Equipment Needs
- Rudolph Autopol III automatic polarimeter
- 10 dm polarimeter cell
- plastic squirt bottle filled with water to rinse cell
- plastic squirt bottle filled with 95% ethanol to rinse cell
Tips for Success
The spots will visualize optimally if the plate is held over the hot plate rather than placed on it
(the TLC plates will darken too rapidly to be useful if the TLC plate is left on the hot plate)
CAS registry numbers
(+)-menthol
18479-68-0
(-)-menthol
2216-51-5
(+)-isomenthol
23283-97-8
(+)-neomenthol 2216-52-6
ethanol
64-17-5
vanillin
121-33-5
hexanes
73513-42-5
sulfuric acid
7664-93-9
ethyl acetate
141-78-6
Safety and Hazards
The students should be carefully not to come in contact with the visualization solution, and
immediately wash any area that comes in contact with the solution thoroughly with water. Inhalation of
the TLC solvent should be avoided.
1. Experimental Organic Chemistry. Practice and Principles. Harwood, L. M.; Moody, C. J.
Blackwell Science Ltd: Oxford, 1989; p 164.
TLC plate
4:1
hexanes:EtOAc
M = (+)-menthol
I = isomenthol
N = neomenthol
M
I
N
(-)-menthol
1:1 mixture of
(+) and (-)-menthol
(+)-menthol
Picture of mixed melting point demonstration.
(The compounds were placed on a preheated piece of colored paper, instead of a
glass petri dish, to avoid glare from flash)
In the Laboratory
ously reported (c 10, EtOH).4 A sample of (+)-menthol at a
concentration of 0.0027 gmL gave a reproducible specific
rotation of +50⬚, while a sample of (−)-menthol at a concentration of 0.0030 gmL gave a reproducible specific rotation of ᎑47⬚.
An intriguing demonstration of how enantiomers have
the same physical properties until placed in chiral environments (thus forming diastereomeric associations) involved
preparing a 1:1 by weight mixed sample of (+)-menthol and
(−)-menthol and observing the mixed melting point. The
melting point was indeed depressed in the mixed sample (mp
= 28.3 − 35.0 ⬚C), and this can be remarkably demonstrated
without the use of a melting point apparatus. If small, wellground samples of (+)-menthol and (−)-menthol are placed
apart on a student’s gloved hand, the enantiomers remain a
solid. Upon mixing, however, the samples quickly begin to
melt and soon no solid material is left.
Alternatively, this can easily be demonstrated to a large
group.5 A glass crystallizing dish is preheated by holding it
an inch from the exhaust port of a typical overhead projector for ca. 30 seconds. The warm dish is then placed on the
writing surface of the projector, and two separate piles of
finely ground (−)-menthol and (+)-menthol are added. After a minute, the piles are mixed using a spatula and the material begins to clump and then melt literally in front of the
audience’s eyes.
Lastly, we had the students identify the chiral centers
and assign the absolute stereochemistry for each compound.
This exercise is particularly rewarding in that the assigning
the priorities at the C-5 carbon it is necessary to proceed
through several bonds to find the first point of difference, as
well as the structures including a methine that is not a stereocenter (the central carbon of the isopropyl group). The concept that enantiomers have all the stereocenters inverted while
diastereomers have some but not all of the stereocenters inverted is also reinforced in this exercise as the students attempt to assign the stereochemistry of all four compounds.
of the stereoisomers being far enough apart to give unequivocal results.
In the reports, a large majority of the students clearly
stated that diastereomers have different physical properties,
while enantiomers only had different physical properties in
chiral environments. Having the students assign the stereochemistry gave them practice at assigning priorities to groups
where the first point of difference is not directly at the chiral
center. All the students were able to assign the C-1 stereocenter, and about 80% correctly assigned the C-2 and C-5
stereocenters. Most students also quickly realized that after
assigning the stereochemistry of the first compound, they only
need to focus on the chiral centers that have changed in the
remaining structures. They then realized that if the stereochemistry has switched, the assignment is simply reversed
[(R) to (S ) or (S ) to (R)]. Another nice facet of these compounds is that there are two different diastereomers used, dispelling the mistaken conceptions that (i) a compound can
only have one enantiomer and one pair of diastereomers and
(ii) that all diastereomers of a compound behave identically
– that since both (+)-neomenthol and (+)-isomenthol are diastereomers of (+)-menthol they should be no difference between the behavior of isomenthol and neomenthol.
The four commercially-available menthol stereoisomers
are ideally suited for the illustration of the differences in physical behavior between diastereomers and enantiomers in either
a first-semester organic laboratory experiment, or as demonstrations in an organic lecture course. Additionally, the mixed
melting behavior of the (+)- and (−)-menthol makes for a fun
and interesting demonstration of a principle that is often neglected in the discussion of the properties of enantiomers.
W
Supplemental Material
Instructions for the students and notes for the instructor are available in this issue of JCE Online.
Notes
Hazards
The visualization solution contains dilute sulfuric acid
and can cause burns. The components of the developing solvent are flammable and irritants, and prolonged exposure to
hexanes can affect the central nervous system.
Results and Conclusions
We have been using this experiment for the past four
semesters and have found that the students not only enjoy it
but also easily learn the conceptual information we wished
to impart. The effectiveness of the experiment was assessed
by the degree of understanding shown in the student’s written lab reports as well as comments made by the students in
the laboratory. From the TLC analysis, they successfully observe that diastereomers have different physical properties,
as isomenthol, neomenthol, and menthol are clearly resolved
on the plate. In some cases, the menthol and isomenthol spots
were not clearly resolved owing to students applying too much
sample to the plate; usually after a second attempt, a clear
TLC plate can be obtained. The melting point analyses were
unequivocal for most of the students, with the melting points
www.JCE.DivCHED.org
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1. The cis isomer is $19.10g in the 2004–2005 Aldrich
Chemical Company, Inc. catalog.
2. An additional exercise would be to have the students obtain the melting point of the individual enantiomers and observe
their similarity.
3. Alternatively, as suggested by one of the reviewers, (+)-menthol, (−)-menthol, and a co-spot of the two enantiomers could be
chromatographed on this plate to more emphatically demonstrate
that the enantiomers behave identically on silica gel.
4. c given in units of grams per mL. The literature rotation
of (+)-menthol was obtained from the Aldrich catalog.
5. Such a demonstration was used for the organic chemistry
I lecture, which typically numbers around 40–50 students.
Literature Cited
1. (a) Knauer, B. J. Chem. Educ. 1989, 66, 1033–1034. (b)
Solomon, S. J. Chem. Educ. 1989, 66, 436–437. (c) Murov,
S. L.; Pickering, M. J. Chem. Educ. 1973, 50, 74–75.
2. (a) α-phenylethylamine: Ault, A. J. Chem. Educ. 1965, 42,
269. Durieu, V.; Martiat, G.; Vandergeten, M. Ch.; Pirsoul,
F.; Toubeau, F.; Van Camp, A. J. Chem. Educ. 2000, 77, 752–
Vol. 82 No. 7 July 2005
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Journal of Chemical Education
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In the Laboratory
753. (b) ibuprofen: Sen, S. E.; Anliker, K. S. J. Chem. Educ.
1996, 73, 569–572. (c) phenylsuccinic acid: Stephani, R.;
Cesare, V. J. Chem. Educ. 1997, 74, 1226. (d) α-methylbenzyl
acetate (enzymatic): Steca, D.; Arends, I. W. C. E.; Hanefeld,
U. J. Chem. Educ. 2002, 79, 1351–1352. (e) γ-methyl-γbutyrolactone synthesis (enzymatic): Lee, M. J. Chem. Educ.
1998, 75, 217–219. (f ) β-hydroxyester synthesis (enzymatic):
North, M. J. Chem. Educ. 1998, 75, 630–631.
3. (a) bromination of fumaric and maleic acids: Tomsho, J.;
McKee, J.; Zanger, M. J. Chem. Educ. 1999, 76, 73–74. (b)
reduction of 1,3-diphenyl-1,3-propanedione: Deprés, J.-P.;
Morat, C. J. Chem. Educ. 1992, 69, A232–A239. (c) reduc-
1048
Journal of Chemical Education
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tion of benzoin: Rowland, A. T. J. Chem. Educ. 1983, 60,
1084–1085.
4. Gilbert, J. C.; Martin, S. F. Experimental Organic Chemistry:
A Miniscale and Microscale Approach, 2nd ed.; Saunders College Publishing: Fort Worth, TX, 1998; pp 178–181.
5. To the best of our knowledge, there is only one other publication that employs menthol in an introductory stereochemistry experiment; in this instance, (−)-menthone is reduced to
give (−)-menthol and (+)-neomenthol (Barry, J. J. Chem. Educ.
1973, 50, 292).
6. The Merck Index, 13th ed.; Budavari, Susan, Ed.; Merck Research Laboratories: Whitehouse Station, NJ, 2001; p 5862.
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