Isolation and Analysis of Essential Oils from SpicesStephen 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 avariety of experimental techniques to undergraduate students in introductory organicchemistry. Eugenol, anethole, and carvone were extracted from six common spices usingsteam-distillation and diethyl ether as the extraction solvent. Students assessed the purity oftheir spice extract and identified its components via TLC, reverse-phase HPLC, and GCMSanalyses. In addition, students reviewed molecular characteristics and interactions whilediscovering 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

Essential oils are hydrophobic natural product mixturesobtained from odoriferous plants. These are believed toserve a role in the development, reproduction, or protection ofthe plant. The quantity and composition of essential oils varieswith several environmental factors, including plant, growth,harvest, and isolation conditions.16 The extracted oils arecharacteristically fragrant and have use as perfumes, flavorings,insecticides, and other medicinal purposes.713

Steam distillation is a simple, classic method of naturalproduct isolation that avoids prolonged heating and possibledecomposition of the organic compounds.1,1416 This methodhas been applied in many undergraduate laboratory experi-ments, including natural product isolation from clove,17

citrus,18,19 anise,20,21 and other sources.19,2225 Followingdistillation, the distillate is commonly extracted with dichloro-methane. Alternative isolation methods,26,27 such as cold-pressextraction,10 solid-phase extraction,28 supercritical fluid extrac-tion,2931 microwave-assisted distillation,3237 and ultrasound-assisted extraction,38 may also be applied to undergraduatelaboratories.3944

A laboratory experiment was developed that combined theclassic isolation of essential oils by steam-distillation using lesshazardous solvents45 with product analysis by modernchromatographic techniques. The six spices employed wereallspice, clove, anise, fennel, dill, and caraway. The isolatedessential oils of these spices contained eugenol, anethole, orcarvone (Table 1) identified through thin-layer chromatog-raphy (TLC),46,47 reverse-phase high-performance liquidchromatography (RP-HPLC),48 and gas chromatographymass spectrometry (GCMS).49,50The isolation of the essential oils from the spice samples

provided the students an opportunity to analyze the relation-ship between molecular structure, physical properties, and

intermolecular forces. Eugenol, trans-anethole, and carvone arehydrophobic oils and readily dissolve in the organic solvents. Inaccord with the differing functional groups, the molecularcharacteristics (polarity, dipoledipole, or hydrogen bonding)are different, as exemplified by the differing physical properties,retention factors in TLC, and retention times in RP-HPLC andGCMS.TLC and RP-HPLC were utilized to qualitatively analyze the

purity of the natural product extracts. Components of thesample mixtures were separated based upon differingintermolecular forces and attraction to the stationary phaseversus solubility in the mobile phase. The stationary phase forTLC was a polar silicate, whereas for RP-HPLC it was anonpolar bead (C18). Eugenol, trans-anethole, and carvone havediffering interactions with the stationary and mobile phases andthus had different Rf values (TLC) and retention times (RP-

Published: January 25, 2012

Table 1. Essential Oils Isolated from Natural Sources

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HPLC). These techniques do not, however, account forcoelution of contaminates that may give rise to false positiveresults. To address this shortcoming, gas chromatography withinline mass spectrometry provided an additional qualitativeanalysis of the spice extracts. The volatile essential oilcomponents were separated based upon boiling point. GCMS retention times correlated with the higher boiling pointspecies having longer retention time (eugenol 256 C, anethole234 C, carvone 231 C).7 Following EI-MS (electronionization mass spectrometry) analysis and peak matchingthrough NIST Mass Spectral Search, students gained aqualitative and semiquantitative assessment of their spice andthe capability to identify other components within the naturalproduct 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 thesimilarities and differences between the essential oils ofdifferent origins, and (iii) demonstrated to the students theinformation obtained from three common chromatographictechniques.

GENERAL EXPERIMENT AND DISCUSSIONThis experiment was implemented as part of the laboratorysequence in an introductory organic chemistry course. Theexperiment was conducted over two, three-hour laboratorysessions. The first week involved isolation of the essential oil,and the second week involved chromatographic character-ization 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 theninstructed to isolate the essential oil from their spice bysteam-distillation, determine a yield, and determine whichessential oil component (eugenol, anethole, or carvone) theirspice contained. The essential oils isolated from each of thespice extracts were determined through the application of threechromatographic techniques (TLC, RP-HPLC, and GCMS)and verified using authentic standard samples. Class data werecompiled, allowing the students to compare and contrastproperties 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. Theorganic phase was dried (sodium sulfate) and the etherevaporated. The yield of the essential oil was dependent upon anumber of experimental factors, including the spice, the care ingrinding the spice sample, and the extraction technique (Table2).An aliquot of the concentrated essential oil sample was

dissolved in a known volume of methanol for chromatographicanalysis. The methanolic sample and the authentic standard

eugenol, anethole, carvone samples were spotted and eluted(10% ethyl acetate, hexanes) on silica TLC plates. This allowedthe student to decipher which essential oil was isolated fromthe assigned spice and to estimate sample purity. TLC alsoafforded an opportunity to compare and contrast the polarity ofthe three essential oils by their elution on the polar plate with arelatively nonpolar eluent. The identity, purity, and the polarityof the essential oils were further assessed through RP-HPLCanalysis of the methanolic spice and authentic standardsamples. In this technique, the support was a nonpolar C18column, eluted isocratically with a polar solvent (85%methanol, water) and spectral detection at 254 nm. Finally,GCMS analysis of the methanolic extract samples confirmedthe identification (NIST MS Search program) of the essentialoil components in the spices. (Due to the small quantity andpurity of the isolated carvone, the laboratory experiment didnot include the determination of optical rotation for thesesamples.)The application of these chromatographic techniques within

one laboratory experiment allowed the students to gain anappreciation for the data acquired from each technique whileillustrating 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 theconstitutive mixtures were separated based upon differences inmolecular interactions with the stationary (silica or C18) andmobile phases. TLC was a fast and simple means of assessingthe samples purity. Only those compounds detected absorbedshortwave UV light or were reactive with iodine. Likewise,reverse-phase HPLC provided a simple and nondestructivequalitative analysis of the samples. Only those elutedcompounds that absorbed 254 nm light were detected. Theorder of compound elution was opposite in RP-HPLC andTLC, in accord with the differing stationary (and mobile) phasepolarity. It was determined that eugenol was the most polar,anethole was the least polar, and carvone had intermediatepolarity. Without further optimization of the chromatographicconditions for each spice, compounds of similar polarity withinthe samples were not resolved and eluted together. This wasdemonstrated experimentally when comparing the RP-HPLCand GCMS data for fennel, caraway, and dill.GCMS analysis detected more components within the

extract samples than the other two techniques and was basedupon the vaporization and ionization of the molecules withinthe methanolic samples. Sample components that were notvolatile under the experimental conditions were not detected.Accordingly, eugenol was identified through GCMS as themajor component of the allspice and clove samples, whileanethole was the major component for the anise sample.Carvone, however, was not the major isolated component ofthe dill or caraway samples. Apiol and fenuron (an herbicide)were the major components of these spice samples,

Table 2. Summary of Representative Essential Oil Data

SpiceAv Mass of Isolated

Oil/g Essential OilTLC Retention Factor,

RfReverse-Phase HPLC Retention Time,

Rt/minGCMS Retention Time,

Rt/min

Allspice 0.29 Eugenol 0.25 3.4 10.2Clove 0.55Anise 0.09 trans-Anethole 0.64 5.7 9.3Fennel 0.06Caraway 0.08 (+)-Carvone 0.47 3.9 8.6Dill 0.06

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respectively. The major detected component of the fennelextract was estragol (an isomer of anethole) rather thananethole. The apiol, fenuron, and estragol matches identified bythe NIST MS Search program were not verified using authenticsamples.

HAZARDSStudents wore standard protective eyewear and gloves. Cautionshould be advised to not allow the distillation to proceed todryness in the heated flask. Exposure to UV lights should belimited, 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 extractsare irritants. Diethyl ether is a highly flammable and volatileliquid that may form peroxides upon storage. It is also harmfulby inhalation, ingestion, or skin contact, causing irritation,dizziness, drowsiness or unconsciousness with prolongedexposure. Methanol is a flammable, volatile liquid and is toxicif ingested. Ethyl acetate and hexane are volatile, flammableliquids that are also irritants. Iodine is corrosive and an irritantthat readily absorbs through the skin.

SUMMARYThe experiment enhanced the students knowledge of naturalproducts and connected organic chemistry to the real world.Students were introduced to natural product isolationtechniques, three chromatographic analyses, and reviewedfundamental molecular characteristics and interactions.Through analysis of the chromatographic data, students wereable to determine which essential oil the assigned spicecontained, relate structure to molecular properties and boilingpoint, rank the relative polarity of the essential oils, and discussthe similarities among the different spice extracts. TLC and RP-HPLC provided a qualitative analysis of the spice extractsamples. The separation of sample components depended uponthe interactions (intermolecular forces) between the samplemolecules and the stationary/mobile phases. The strength ofthe interactions was related to the molecular structure(functional groups) of the essential oil components. GCMSanalysis provided qualitative data and identification of the spiceextract components. The separation of sample components wasaccording to the boiling point (vaporization). Students wereable to discover similarities among the different spices throughthe application of these chromatographic methods: allspice andclove contained eugenol; anise and fennel contained trans-anethole; and caraway and dill contained carvone.

ASSOCIATED CONTENT*S Supporting InformationExperimental handout for students; notes for the instructor;representative student data. This material is available via theInternet at http://pubs.acs.org.

AUTHOR INFORMATIONCorresponding Author

*E-mail: lrossi@rwu.edu.

ACKNOWLEDGMENTSWe acknowledge the laboratory instructors and studentsenrolled in the Organic Chemistry I and II courses at RogerWilliams University for their many contributions toward the

development and implementation of this laboratory experi-ment.

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