Laboratory Experiment

pubs.acs.org/jchemeduc

Isolation and Analysis of Essential Oils from Spices

Stephen K. O’Shea,* 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 GC−MS
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

E 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
Table 1. Essential Oils Isolated from Natural Sources

the plant. The quantity and composition of essential oils varies

with several environmental factors, including plant, growth,
harvest, and isolation conditions.1−6 The extracted oils are
characteristically fragrant and have use as perfumes, flavorings,
insecticides, and other medicinal purposes.7−13
Steam distillation is a simple, classic method of natural
product isolation that avoids prolonged heating and possible
decomposition of the organic compounds.1,14−16 This method
has been applied in many undergraduate laboratory experi-
ments, including natural product isolation from clove,17
citrus,18,19 anise,20,21 and other sources.19,22−25 Following
distillation, the distillate is commonly extracted with dichloro- intermolecular forces. Eugenol, trans-anethole, and carvone are
methane. Alternative isolation methods,26,27 such as cold-press hydrophobic oils and readily dissolve in the organic solvents. In
extraction,10 solid-phase extraction,28 supercritical fluid extrac- accord with the differing functional groups, the molecular
tion,29−31 microwave-assisted distillation,32−37 and ultrasound- characteristics (polarity, dipole−dipole, or hydrogen bonding)
assisted extraction,38 may also be applied to undergraduate are different, as exemplified by the differing physical properties,
laboratories.39−44 retention factors in TLC, and retention times in RP-HPLC and
A laboratory experiment was developed that combined the GC−MS.
classic isolation of essential oils by steam-distillation using less TLC and RP-HPLC were utilized to qualitatively analyze the
hazardous solvents45 with product analysis by modern
purity of the natural product extracts. Components of the
chromatographic techniques. The six spices employed were
sample mixtures were separated based upon differing
allspice, clove, anise, fennel, dill, and caraway. The isolated
intermolecular forces and attraction to the stationary phase
essential oils of these spices contained eugenol, anethole, or
carvone (Table 1) identified through thin-layer chromatog- versus solubility in the mobile phase. The stationary phase for
raphy (TLC),46,47 reverse-phase high-performance liquid TLC was a polar silicate, whereas for RP-HPLC it was a
chromatography (RP-HPLC),48 and gas chromatography− nonpolar bead (C18). Eugenol, trans-anethole, and carvone have
mass spectrometry (GC−MS).49,50 differing interactions with the stationary and mobile phases and
The isolation of the essential oils from the spice samples thus had different Rf values (TLC) and retention times (RP-
provided the students an opportunity to analyze the relation-
ship between molecular structure, physical properties, and Published: January 25, 2012
© 2012 American Chemical Society and
Division of Chemical Education, Inc. 665 dx.doi.org/10.1021/ed101141w | J. Chem. Educ. 2012, 89, 665−668
Journal of Chemical Education Laboratory Experiment

Table 2. Summary of Representative Essential Oil Data

Av Mass of Isolated TLC Retention Factor, Reverse-Phase HPLC Retention Time, GC−MS Retention Time,
Spice Oil/g Essential Oil Rf Rt/min Rt/min
Allspice 0.29 Eugenol 0.25 3.4 10.2
Clove 0.55
Anise 0.09 trans-Anethole 0.64 5.7 9.3
Fennel 0.06
Caraway 0.08 (+)-Carvone 0.47 3.9 8.6
Dill 0.06

HPLC). These techniques do not, however, account for eugenol, anethole, carvone samples were spotted and eluted
coelution of contaminates that may give rise to false positive (10% ethyl acetate, hexanes) on silica TLC plates. This allowed
results. To address this shortcoming, gas chromatography with the student to decipher which essential oil was isolated from
inline mass spectrometry provided an additional qualitative the assigned spice and to estimate sample purity. TLC also
analysis of the spice extracts. The volatile essential oil afforded an opportunity to compare and contrast the polarity of
components were separated based upon boiling point. GC− the three essential oils by their elution on the polar plate with a
MS retention times correlated with the higher boiling point relatively nonpolar eluent. The identity, purity, and the polarity
species having longer retention time (eugenol 256 °C, anethole of the essential oils were further assessed through RP-HPLC
234 °C, carvone 231 °C).7 Following EI-MS (electron analysis of the methanolic spice and authentic standard
ionization mass spectrometry) analysis and peak matching samples. In this technique, the support was a nonpolar C18
through NIST Mass Spectral Search, students gained a column, eluted isocratically with a polar solvent (85%
qualitative and semiquantitative assessment of their spice and methanol, water) and spectral detection at 254 nm. Finally,
the capability to identify other components within the natural GC−MS analysis of the methanolic extract samples confirmed
the identification (NIST MS Search program) of the essential
product extract (Table 2).
oil components in the spices. (Due to the small quantity and
The laboratory experiment described herein (i) introduced
students to steam-distillation and extraction techniques; (ii) purity of the isolated carvone, the laboratory experiment did
promoted collaboration among the students to determine the not include the determination of optical rotation for these
similarities and differences between the essential oils of samples.)
different origins, and (iii) demonstrated to the students the The application of these chromatographic techniques within
information obtained from three common chromatographic one laboratory experiment allowed the students to gain an
techniques. appreciation for the data acquired from each technique while

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illustrating some of the benefits and limitations of each. Overall,
GENERAL EXPERIMENT AND DISCUSSION only a small organic sample was required for the three analyses.
In the RP-HPLC and TLC techniques, components of the
This experiment was implemented as part of the laboratory
sequence in an introductory organic chemistry course. The constitutive mixtures were separated based upon differences in
experiment was conducted over two, three-hour laboratory molecular interactions with the stationary (silica or C18) and
sessions. The first week involved isolation of the essential oil, mobile phases. TLC was a fast and simple means of assessing
and the second week involved chromatographic character- the sample’s purity. Only those compounds detected absorbed
ization of the extractant. shortwave UV light or were reactive with iodine. Likewise,
Undergraduate students in each organic chemistry laboratory reverse-phase HPLC provided a simple and nondestructive
section were assigned one of six different spices (allspice, clove, qualitative analysis of the samples. Only those eluted
anise, fennel, dill, or caraway). The students were then compounds that absorbed 254 nm light were detected. The
instructed to isolate the essential oil from their spice by order of compound elution was opposite in RP-HPLC and
steam-distillation, determine a yield, and determine which TLC, in accord with the differing stationary (and mobile) phase
essential oil component (eugenol, anethole, or carvone) their polarity. It was determined that eugenol was the most polar,
spice contained. The essential oils isolated from each of the anethole was the least polar, and carvone had intermediate
spice extracts were determined through the application of three polarity. Without further optimization of the chromatographic
chromatographic techniques (TLC, RP-HPLC, and GC−MS) conditions for each spice, compounds of similar polarity within
and verified using authentic standard samples. Class data were the samples were not resolved and eluted together. This was
compiled, allowing the students to compare and contrast demonstrated experimentally when comparing the RP-HPLC
properties exhibited by each spice and essential oil component. and GC−MS data for fennel, caraway, and dill.
The essential oils were isolated by the steam-distillation of an GC−MS analysis detected more components within the
aqueous slurry containing the ground spice sample (10−15 g). extract samples than the other two techniques and was based
The steam-distillate was back extracted into diethyl ether. The upon the vaporization and ionization of the molecules within
organic phase was dried (sodium sulfate) and the ether the methanolic samples. Sample components that were not
evaporated. The yield of the essential oil was dependent upon a volatile under the experimental conditions were not detected.
number of experimental factors, including the spice, the care in Accordingly, eugenol was identified through GC−MS as the
grinding the spice sample, and the extraction technique (Table major component of the allspice and clove samples, while
2). anethole was the major component for the anise sample.
An aliquot of the concentrated essential oil sample was Carvone, however, was not the major isolated component of
dissolved in a known volume of methanol for chromatographic the dill or caraway samples. Apiol and fenuron (an herbicide)
analysis. The methanolic sample and the authentic standard were the major components of these spice samples,
666 dx.doi.org/10.1021/ed101141w | J. Chem. Educ. 2012, 89, 665−668
Journal of Chemical Education Laboratory Experiment

respectively. The major detected component of the fennel development and implementation of this laboratory experi-
extract was estragol (an isomer of anethole) rather than ment.

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anethole. The apiol, fenuron, and estragol matches identified by
the NIST MS Search program were not verified using authentic REFERENCES
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