In the Laboratory
Analysis of Peppermint Leaf and Spearmint Leaf Extracts by Thin-Layer Chromatography Libbie S. W. Pelter,* Andrea Amico, Natalie Gordon, Chylah Martin, Dessalyn Sandifer, and Michael W. Pelter Department of Chemistry and Physics, Purdue University Calumet, Hammond, IN 46323-2094; *
[email protected] Thin-layer chromatography (TLC) has been an important analytical tool for many years (1). Recently, TLC was described as “The ‘Eyes’ of the Organic Chemist” (2). In this inquirybased activity, the usefulness of TLC to visualize the difference between spearmint and peppermint is explored. The experiment, an adaptation of an earlier report (3), may be used in any class where TLC is discussed from high school to college level. We have used this activity with science majors in an organic chemistry laboratory, with non-science majors in a brewing science class, and in a general science class for elementary education majors. The experiment can be completed in a two-hour period. Both spearmint (Mentha spicata) and peppermint (Mentha piperita) are common plants in the United States and Canada (4). Peppermint is approved for medicinal use and, like spearmint, is used in the flavor and fragrance industry (5). The essential oils from these plants are obtained through steam distillation of the fresh flowering plants. The characteristic chemical constituents of these oils are largely monoterpenes. The chief constituent of spearmint oil is (R)-(−)-carvone (Figure 1), which occurs to the extent of 45 to 60 percent (5, 6). A major constituent of peppermint oil is (1R,2S,5R)-(−)-menthol, which is absent in spearmint oil. Peppermint oil also includes other terpenes including menthone (10–40%), menthyl acetate (4–10%), and menthofuran (0–10%) (Figure 2) (5, 7). Limonene and cineole are also found in significant concentrations (1–20%) in both spearmint oil and peppermint oil (Figure 3). Depending on the source, peppermint oil typically contains between 50 and 85% menthol and must contain 30–55% menthol and less than 1.0% carvone to be considered European pharmacopeial grade (5). The terpenes, (R)-(−)-carvone and (1R,2S,5R)-(−)-menthol, are responsible for cool and minty sensations, but their enantiomers have distinctly different flavor and fragrance characteristics. A comparison of the odor characteristics of these compounds with their stereoisomers provides background for a good discussion on structure–activity relationships (8). After a general explanation of the TLC technique, the students are asked to determine whether this method could be useful for differentiation of spearmint oil and peppermint oil. Students are provided with dilute samples of carvone, menthol, spearmint oil, and peppermint oil for spotting on TLC plates. A developing chamber is also provided with an appropriate developing solvent (10% ethyl acetate in hexane). Two visualization methods are also provided, a UV light and anisaldehyde solution (9). Students are given instruction on the use of each method and cautioned to use the UV light for visualization prior to dipping the developed plates in anisaldehyde solution. Students determine that an effective experimental method is to spot a TLC plate with both known components and one of the oils.
CH3 O
H
CH3
H2C
(R)-(–)carvone
Figure 1. The principal constituent of spearmint oil.
H
H
CH3
CH3
OH H
O
H
H
(1R, 2S, 5R)(–)-menthol
menthone H
CH3 OAc H
H3C
O
H CH3
menthofuran
menthyl acetate
Figure 2. The principal constituents of peppermint oil.
CH3
CH3
O H3C
CH3
1,8-cineole
H3C
CH2
limonene
Figure 3. Terpenes present in both spearmint and peppermint oils.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 1 January 2008 • Journal of Chemical Education
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In the Laboratory
Hazards All of the chemicals and reagents used in the experiment are flammable. The TLC visualization stain is corrosive. Silica gel should not be inhaled. Students should wear safety goggles and gloves. Direct viewing of the UV lamp is harmful to the eyes and should be avoided. It is recommended that UVEX goggles be worn during UV visualization to protect the eyes from UV radiation.
Sample
Results Typical student results are shown in Table 1. Carvone is visible under UV light but menthol is not. This causes some concern with the students, and they must be reminded to develop their TLC plate with the anisaldehyde solution before they conclude the experiment. Upon treatment with the anisaldehyde solution, the menthol spot becomes visible as a blue-colored spot. The spots corresponding to carvone turn yellow upon treatment with the anisaldehyde solution. In the peppermint extract, there may be several spots present, in addition to the menthol. These are the menthone, menthyl acetate, and limonene. These compounds are available and spotting solutions could be provided for each possible compound in this experiment. We found that students were more successful when the experiment was simplified to include only the major constituents. Limonene was provided for students in some experiments, because it was present in both spearmint and peppermint oil (Rf value 0.94–0.98).
Carvone Spearmint extract
Table 1. Student Results from TLC Number of Spots Visible Rf Value(s) Under UV After Treatment Light with Anisaldehyde 0.5–0.6 1 1 yellow 0.5–0.6
1
1 yellow
Peppermint extract
0.35–0.4 0.6 0.95
2
2 blue 1 green
Menthol
0.35–0.4
None
1 blue
Literature Cited
While most students are able to recognize spearmint and peppermint from their distinctive aromas, this experiment allows them to visualize the differences. Calculation of the Rf values for carvone and menthol allows the students to verify their presence in the respective extracts. The use of the anisaldehyde solution allows for the menthol to be clearly seen, and the respective color of the spots clearly shows the difference.
1. Williams, K. R. J. Chem. Educ. 2002, 79, 922. 2. Dickson, H.; Kittredge, K. W.; Sarquis, A. M. J. Chem. Educ. 2004, 81, 1023. 3. Jaspersen-Schib, R. Pharm. Acta Helv. 1961, 36, 141. 4. Sievers, A. F. The Herb Hunters Guide. Misc. Publ. No. 77. USDA: Washington DC, 1930. http://www.hort.purdue.edu/ newcrop/HerbHunters/hhunters.html (accessed Sep 2007). 5. (a) American Botanical Council. The Complete German Commission E Monographs: Therapeutic Guide to Herbal Medicines, 1999. http:// www.herbalgram.org (accessed Sep 2007). (b) European Directorate for the Quality of Medicines. European Pharmacopoeia, 2006. http://www. pheur.org (accessed Sep 2007). (c) U. S. Pharmacopoeia. United States Pharmacopeia–National Formulary (USP–NF), 2007. http://www.usp. org (accessed Sep 2007). (d) Council of the Pharmaceutical Society of Great Britain. The British Pharmaceutical Codex, 1911. http://www. henriettesherbal.com/eclectic/index.html (accessed Sep 2007). 6. (a) Davies, D. R.; Johnson, T. M. J. Chem. Educ. 2007, 84, 318. (b) Dìaz-Maroto, M. C.; Pérez-Coello, M. S.; Viñas, M. A. G.; Cabezudo, M. D. J. Agric. Food Chem. 2003, 51, 1265. 7. Rohloff, J. J. Agric. Food Chem. 1999, 47, 3782. 8. Kimbrough, D. R. J. Chem. Educ. 1997, 74, 861. 9. (a) Gordon, A. J.; Ford, R. A. The Chemist’s Companion; WileyInterscience: New York, 1972; p 379. (b) Nash, J. J.; Meyer, J. A.; Everson, B. J. Chem. Educ. 2001, 78, 364.
Acknowledgments
Supporting JCE Online Material
Conclusions
Financial support was provided by the Louis Stokes Alliance for Minority Participation (LSAMP) Indiana (summer stipends for AA, NG, CM, and DS) and by the Purdue University Calumet Undergraduate Research Program. We thank the students in SCI 150 Brewing Science and SCI 113 Introduction to Physical Science II for their assistance in field-testing the procedure.
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