Laboratory Experiment

pubs.acs.org/jchemeduc

Isolation and Analysis of Essential Oils from Spices

Stephen K. OShea,* Daniel D. Von Riesen,* and Lauren L. Rossi*
Department of Chemistry, Roger Williams University, Bristol, Rhode Island 02809, United States
S Supporting Information
*

ABSTRACT: Natural product isolation and analysis provide an opportunity to present a

variety of experimental techniques to undergraduate students in introductory organic
chemistry. Eugenol, anethole, and carvone were extracted from six common spices using
steam-distillation and diethyl ether as the extraction solvent. Students assessed the purity of
their spice extract and identified its components via TLC, reverse-phase HPLC, and GCMS
analyses. In addition, students reviewed molecular characteristics and interactions while
discovering the similarities and differences among the six spice extracts.

KEYWORDS: Second-Year Undergraduate, Laboratory Instruction, Organic Chemistry, Hands-On Learning/Manipulatives,

Chromatography, Mass Spectrometry, NMR Spectroscopy, Natural Products, Plant Chemistry

Table 1. Essential Oils Isolated from Natural Sources

ssential oils are hydrophobic natural product mixtures

obtained from odoriferous plants. These are believed to
serve a role in the development, reproduction, or protection of
the plant. The quantity and composition of essential oils varies
with several environmental factors, including plant, growth,
harvest, and isolation conditions.16 The extracted oils are
characteristically fragrant and have use as perfumes, flavorings,
insecticides, and other medicinal purposes.713
Steam distillation is a simple, classic method of natural
product isolation that avoids prolonged heating and possible
decomposition of the organic compounds.1,1416 This method
has been applied in many undergraduate laboratory experiments, including natural product isolation from clove,17
citrus,18,19 anise,20,21 and other sources.19,2225 Following
distillation, the distillate is commonly extracted with dichloromethane. Alternative isolation methods,26,27 such as cold-press
extraction,10 solid-phase extraction,28 supercritical fluid extraction,2931 microwave-assisted distillation,3237 and ultrasoundassisted extraction,38 may also be applied to undergraduate
laboratories.3944
A laboratory experiment was developed that combined the
classic isolation of essential oils by steam-distillation using less
hazardous solvents45 with product analysis by modern
chromatographic techniques. The six spices employed were
allspice, clove, anise, fennel, dill, and caraway. The isolated
essential oils of these spices contained eugenol, anethole, or
carvone (Table 1) identified through thin-layer chromatography (TLC),46,47 reverse-phase high-performance liquid
chromatography (RP-HPLC),48 and gas chromatography
mass spectrometry (GCMS).49,50
The isolation of the essential oils from the spice samples
provided the students an opportunity to analyze the relationship between molecular structure, physical properties, and
2012 American Chemical Society and
Division of Chemical Education, Inc.

intermolecular forces. Eugenol, trans-anethole, and carvone are

hydrophobic oils and readily dissolve in the organic solvents. In
accord with the differing functional groups, the molecular
characteristics (polarity, dipoledipole, or hydrogen bonding)
are different, as exemplified by the differing physical properties,
retention factors in TLC, and retention times in RP-HPLC and
GCMS.
TLC and RP-HPLC were utilized to qualitatively analyze the
purity of the natural product extracts. Components of the
sample mixtures were separated based upon differing
intermolecular forces and attraction to the stationary phase
versus solubility in the mobile phase. The stationary phase for
TLC was a polar silicate, whereas for RP-HPLC it was a
nonpolar bead (C18). Eugenol, trans-anethole, and carvone have
differing interactions with the stationary and mobile phases and
thus had different Rf values (TLC) and retention times (RPPublished: January 25, 2012
665

dx.doi.org/10.1021/ed101141w | J. Chem. Educ. 2012, 89, 665668

Journal of Chemical Education

Laboratory Experiment

Table 2. Summary of Representative Essential Oil Data

Spice

Av Mass of Isolated
Oil/g

Allspice
Clove
Anise
Fennel
Caraway
Dill

0.29
0.55
0.09
0.06
0.08
0.06

TLC Retention Factor,

Rf

Reverse-Phase HPLC Retention Time,

Rt/min

GCMS Retention Time,

Rt/min

Eugenol

0.25

3.4

10.2

trans-Anethole

0.64

5.7

9.3

(+)-Carvone

0.47

3.9

8.6

Essential Oil

eugenol, anethole, carvone samples were spotted and eluted

(10% ethyl acetate, hexanes) on silica TLC plates. This allowed
the student to decipher which essential oil was isolated from
the assigned spice and to estimate sample purity. TLC also
afforded an opportunity to compare and contrast the polarity of
the three essential oils by their elution on the polar plate with a
relatively nonpolar eluent. The identity, purity, and the polarity
of the essential oils were further assessed through RP-HPLC
analysis of the methanolic spice and authentic standard
samples. In this technique, the support was a nonpolar C18
column, eluted isocratically with a polar solvent (85%
methanol, water) and spectral detection at 254 nm. Finally,
GCMS analysis of the methanolic extract samples confirmed
the identification (NIST MS Search program) of the essential
oil components in the spices. (Due to the small quantity and
purity of the isolated carvone, the laboratory experiment did
not include the determination of optical rotation for these
samples.)
The application of these chromatographic techniques within
one laboratory experiment allowed the students to gain an
appreciation for the data acquired from each technique while
illustrating some of the benefits and limitations of each. Overall,
only a small organic sample was required for the three analyses.
In the RP-HPLC and TLC techniques, components of the
constitutive mixtures were separated based upon differences in
molecular interactions with the stationary (silica or C18) and
mobile phases. TLC was a fast and simple means of assessing
the samples purity. Only those compounds detected absorbed
shortwave UV light or were reactive with iodine. Likewise,
reverse-phase HPLC provided a simple and nondestructive
qualitative analysis of the samples. Only those eluted
compounds that absorbed 254 nm light were detected. The
order of compound elution was opposite in RP-HPLC and
TLC, in accord with the differing stationary (and mobile) phase
polarity. It was determined that eugenol was the most polar,
anethole was the least polar, and carvone had intermediate
polarity. Without further optimization of the chromatographic
conditions for each spice, compounds of similar polarity within
the samples were not resolved and eluted together. This was
demonstrated experimentally when comparing the RP-HPLC
and GCMS data for fennel, caraway, and dill.
GCMS analysis detected more components within the
extract samples than the other two techniques and was based
upon the vaporization and ionization of the molecules within
the methanolic samples. Sample components that were not
volatile under the experimental conditions were not detected.
Accordingly, eugenol was identified through GCMS as the
major component of the allspice and clove samples, while
anethole was the major component for the anise sample.
Carvone, however, was not the major isolated component of
the dill or caraway samples. Apiol and fenuron (an herbicide)
were the major components of these spice samples,

HPLC). These techniques do not, however, account for

coelution of contaminates that may give rise to false positive
results. To address this shortcoming, gas chromatography with
inline mass spectrometry provided an additional qualitative
analysis of the spice extracts. The volatile essential oil
components were separated based upon boiling point. GC
MS retention times correlated with the higher boiling point
species having longer retention time (eugenol 256 C, anethole
234 C, carvone 231 C).7 Following EI-MS (electron
ionization mass spectrometry) analysis and peak matching
through NIST Mass Spectral Search, students gained a
qualitative and semiquantitative assessment of their spice and
the capability to identify other components within the natural
product extract (Table 2).
The laboratory experiment described herein (i) introduced
students to steam-distillation and extraction techniques; (ii)
promoted collaboration among the students to determine the
similarities and differences between the essential oils of
different origins, and (iii) demonstrated to the students the
information obtained from three common chromatographic
techniques.

GENERAL EXPERIMENT AND DISCUSSION

This experiment was implemented as part of the laboratory
sequence in an introductory organic chemistry course. The
experiment was conducted over two, three-hour laboratory
sessions. The first week involved isolation of the essential oil,
and the second week involved chromatographic characterization of the extractant.
Undergraduate students in each organic chemistry laboratory
section were assigned one of six different spices (allspice, clove,
anise, fennel, dill, or caraway). The students were then
instructed to isolate the essential oil from their spice by
steam-distillation, determine a yield, and determine which
essential oil component (eugenol, anethole, or carvone) their
spice contained. The essential oils isolated from each of the
spice extracts were determined through the application of three
chromatographic techniques (TLC, RP-HPLC, and GCMS)
and verified using authentic standard samples. Class data were
compiled, allowing the students to compare and contrast
properties exhibited by each spice and essential oil component.
The essential oils were isolated by the steam-distillation of an
aqueous slurry containing the ground spice sample (1015 g).
The steam-distillate was back extracted into diethyl ether. The
organic phase was dried (sodium sulfate) and the ether
evaporated. The yield of the essential oil was dependent upon a
number of experimental factors, including the spice, the care in
grinding the spice sample, and the extraction technique (Table
2).
An aliquot of the concentrated essential oil sample was
dissolved in a known volume of methanol for chromatographic
analysis. The methanolic sample and the authentic standard
666

dx.doi.org/10.1021/ed101141w | J. Chem. Educ. 2012, 89, 665668

Journal of Chemical Education

Laboratory Experiment

development and implementation of this laboratory experiment.

respectively. The major detected component of the fennel

extract was estragol (an isomer of anethole) rather than
anethole. The apiol, fenuron, and estragol matches identified by
the NIST MS Search program were not verified using authentic
samples.

(1) Guenther, E. The Essential Oils; D.Van Nostrand Company Inc.:

New York, 1948; Vol. I.
(2) Fraenkel, G. S. Science 1959, 129 (3361), 14661470.
(3) Berenbaum, M. R.; Zangerl, A. R. Plant Physiol. 2008, 146 (3),
804811.
(4) Putz, A. Proksch, P. In Annual Plant Reviews Vol. 39: Functions
and Biotechnology of Plant Secondary Metabolites, 2nd ed.; Wink, M.,
Ed.; Wiley-Blackwell: Oxford, U.K., 2010; pp 162213.
(5) Reichling, J. In Annual Plant Reviews Vol. 39: Functions and
Biotechnology of Plant Secondary Metabolites, 2nd ed.; Wink, M., Ed.;
Wiley-Blackwell: Oxford, U.K., 2010; pp 214347.
(6) Wink, M. Schimmer, O. In Annual Plant Reviews Vol. 39:
Functions and Biotechnology of Plant Secondary Metabolites, 2nd ed.;
Wink, M., Ed.; Wiley-Blackwell: Oxford, U.K., 2010; pp 21161.
(7) Guenther, E. The Essential Oils; D. Van Nostrand Company, Inc.:
New York, 1949; Vol. II.
(8) Kalemba, D.; Kunicka, A. Curr. Med. Chem. 2003, 10 (10), 813
829.
(9) Burt, S. Int. J. Food Microbiol. 2004, 94 (3), 223253.
(10) Scott, R. P. W. In Encyclopedia of Analytical Science; Paul, W.,
Alan, T. Colin, P., Eds.; Elsevier: Oxford, 2005; pp 554561.
(11) Bakkali, F.; Averbeck, S.; Averbeck, D.; Idaomar, M. Food Chem.
Toxicol. 2008, 46 (2), 446475.
(12) Baby, S. George, V. In New Strategies Combating Bacterial
Infection; Wiley-VCH Verlag GmbH & Co. KGaA: 2009; pp 165203.
(13) Antunes, M. D. C.; Cavaco, A. M. Flavour Fragance J. 2010, 25,
351366.
(14) Clevenger, J. F. Am. Perfum. Essent. Oil Rev. 1928, 23, 467503.
(15) Franklin, W. J.; Keyzer, H. Anal. Chem. 1962, 34 (12), 1650
1653.
(16) Dix, K. D.; Fritz, J. S. J. Chromatogr., A 1987, 408, 201210.
(17) Ntamila, M. S.; Hassanali, A. J. Chem. Educ. 1976, 53 (4), 263.
(18) Greenberg, F. H. J. Chem. Educ. 1968, 45 (8), 537538.
(19) Glidewell, C. J. Chem. Educ. 1991, 68 (3), 267269.
(20) LeFevre, J. W. J. Chem. Educ. 2000, 77 (3), 361363.
(21) Garin, D. L. J. Chem. Educ. 1980, 57 (2), 138.
(22) Runquist, O. J. Chem. Educ. 1969, 46 (12), 846847.
(23) Griffin, R. W. J. Chem. Educ. 1974, 51 (9), 601602.
(24) Garin, D. L. J. Chem. Educ. 1976, 53 (2), 105.
(25) Craveiro, A. A.; Matos, F. J. A.; de Alencar, J. W. J. Chem. Educ.
1976, 53 (10), 652.
(26) Starmans, D. A. J.; Nijhuis, H. H. Trends Food Sci. Technol. 1996,
7 (6), 191197.
(27) Luque, d. C. M. D.; Jimenez-Carmona, M. M.; Fernandez-Perez,
V. TrAC, Trends Anal. Chem. 1999, 18 (11), 708716.
(28) Richter, J.; Schellenberg, I. Anal. Bioanal. Chem. 2007, 387 (6),
22072217.
(29) Moyler, D. A. Flavour Fragrance J. 1993, 8 (5), 235247.
(30) Reverchon, E.; De Marco, I. J. Supercrit. Fluids 2006, 38 (2),
146166.
(31) Chester, T. L.; Pinkston, J. D.; Raynie, D. E. Anal. Chem. 1996,
68 (12), 487514.
(32) Sparr Eskilsson, C.; Bjorklund, E. J. Chromatogr., A 2000, 902
(1), 227250.
(33) Lucchesi, M. E.; Chemat, F.; Smadja, J. Flavour Fragrance J.
2004, 19 (2), 134138.
(34) Golmakani, M.-T.; Rezaei, K. Food Chem. 2008, 109 (4), 925
930.
(35) Vian, M. A.; Fernandez, X.; Visinoni, F.; Chemat, F. J.
Chromatogr., A 2008, 1190 (12), 1417.
(36) Farhat, A.; Ginies, C.; Romdhane, M.; Chemat, F. J.
Chromatogr., A 2009, 1216 (26), 50775085.
(37) Farhat, A.; Fabiano-Tixier, A.-S.; Visinoni, F.; Romdhane, M.;
Chemat, F. J. Chromatogr., A 2010, 1217 (47), 73457350.

HAZARDS
Students wore standard protective eyewear and gloves. Caution
should be advised to not allow the distillation to proceed to
dryness in the heated flask. Exposure to UV lights should be
limited, as it may cause damage to eyes or skin.
All of the compounds used should be handled in a manner
consistent with the appropriate safety data. Essential oil extracts
are irritants. Diethyl ether is a highly flammable and volatile
liquid that may form peroxides upon storage. It is also harmful
by inhalation, ingestion, or skin contact, causing irritation,
dizziness, drowsiness or unconsciousness with prolonged
exposure. Methanol is a flammable, volatile liquid and is toxic
if ingested. Ethyl acetate and hexane are volatile, flammable
liquids that are also irritants. Iodine is corrosive and an irritant
that readily absorbs through the skin.

SUMMARY
The experiment enhanced the students knowledge of natural
products and connected organic chemistry to the real world.
Students were introduced to natural product isolation
techniques, three chromatographic analyses, and reviewed
fundamental molecular characteristics and interactions.
Through analysis of the chromatographic data, students were
able to determine which essential oil the assigned spice
contained, relate structure to molecular properties and boiling
point, rank the relative polarity of the essential oils, and discuss
the similarities among the different spice extracts. TLC and RPHPLC provided a qualitative analysis of the spice extract
samples. The separation of sample components depended upon
the interactions (intermolecular forces) between the sample
molecules and the stationary/mobile phases. The strength of
the interactions was related to the molecular structure
(functional groups) of the essential oil components. GCMS
analysis provided qualitative data and identification of the spice
extract components. The separation of sample components was
according to the boiling point (vaporization). Students were
able to discover similarities among the different spices through
the application of these chromatographic methods: allspice and
clove contained eugenol; anise and fennel contained transanethole; and caraway and dill contained carvone.

ASSOCIATED CONTENT

S Supporting Information
*

Experimental handout for students; notes for the instructor;

representative student data. This material is available via the
Internet at http://pubs.acs.org.

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: lrossi@rwu.edu.

ACKNOWLEDGMENTS
We acknowledge the laboratory instructors and students
enrolled in the Organic Chemistry I and II courses at Roger
Williams University for their many contributions toward the
667

dx.doi.org/10.1021/ed101141w | J. Chem. Educ. 2012, 89, 665668

Journal of Chemical Education

Laboratory Experiment

(38) Esclapez, M.; Garca-Perez, J.; Mulet, A.; Carcel, J. Food Eng. Rev
2011, 3 (2), 108120.
(39) Garner, C. M.; Garibaldi, C. J. Chem. Educ. 1994, 71 (6), A146
A147.
(40) Williams, K. R.; Pierce, R. E. J. Chem. Educ. 1998, 75 (2), 223
226.
(41) Galipo, R. C.; Canhoto, A. J.; Walla, M. D.; Morgan, S. L. J.
Chem. Educ. 1999, 76 (2), 245248.
(42) McKenzie, L. C.; Thompson, J. E.; Sullivan, R.; Hutchison, J. E.
Green Chem. 2004, 6 (8), 355358.
(43) Pelletier, A.; Lavoie, J.-M.; Chornet, E. J. Chem. Educ. 2008, 85
(11), 15551557.
(44) Menguy, L.; Prim, D.; Carlin-Sinclair, A.; Marc, I. J. Chem. Educ.
2009, 86 (11), 13071310.
(45) Capello, C.; Fischer, U.; Hungerbuhler, K. Green Chem. 2007, 9
(9), 927934.
(46) Malins, D. C.; Wekell, J. C. J. Chem. Educ. 1963, 40 (10), 531
534.
(47) Lawrence, B. M. Perfum. Essent. Oil Rec. 1968, 59, 421432.
(48) McKone, H. T. J. Chem. Educ. 1979, 56 (10), 698.
(49) Eiceman, G. A.; Hill, H. H.; Davani, B.; Gardea-Torresday, J.
Anal. Chem. 1996, 68 (12), 291308.
(50) Marriott, P. J.; Shellie, R.; Cornwell, C. J. Chromatogr., A 2001,
936 (12), 122.

668

dx.doi.org/10.1021/ed101141w | J. Chem. Educ. 2012, 89, 665668