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
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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.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 experiments, 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 dichloromethane. Alternative isolation methods,26,27 such as cold-press extraction,10 solid-phase extraction,28 supercritical fluid extraction,29−31 microwave-assisted distillation,32−37 and ultrasoundassisted extraction,38 may also be applied to undergraduate laboratories.39−44 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 (GC−MS).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, dipole−dipole, or hydrogen bonding) are different, as exemplified by the differing physical properties, retention factors in TLC, and retention times in RP-HPLC and GC−MS. 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, 665−668
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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
GC−MS 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, GC−MS 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 sample’s 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 GC−MS data for fennel, caraway, and dill. GC−MS 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 GC−MS 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.
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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 GC−MS) 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 (10−15 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
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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.
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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.
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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. GC−MS 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.
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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.
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REFERENCES
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
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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
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