In the Laboratory edited by
The Microscale Laboratory
R. David Crouch Dickinson College Carlisle, PA 17013-2896
Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer Chromatography
W
Don R. Davies* and Todd M. Johnson Department of Chemistry, Weber State University, Ogden, UT 84408; *
[email protected] Column chromatography is one of the most common and effective methods of separating mixtures of organic compounds. Owing to the utility of this technique and its general application to separations technologies, column chromatography is frequently incorporated into undergraduate organic teaching laboratories. There are several reports in the literature of column chromatographic separations of dyes, plant pigments, and other colored compounds specifically designed for students (1–7). However, less common are reports of separations and visualization techniques involving colorless components (8, 9). Since most organic compounds are colorless, chemistry students should be taught indirect methods to visualize the success of a given separation and identify fractions containing similar components (10, 11). The goal of this project was to design a simple, yet representative experiment where students separate a colorless mixture using column chromatography and then monitor the outcome of the separation using thin-layer chromatography (TLC) and infrared spectroscopy (IR). Spearmint oil, a natural product with a pleasant aroma, was chosen as the colorless mixture. Although spearmint oil contains at least 28 different organic compounds (12, 13), only three easily separable components, (+)-limonene, L-(−)carvone and (1R,2R,4R )-dihydrocarveol, are readily visible by permanganate dip, and, of these three components, only L-(−)-carvone is visible under a UV lamp. In 2000, Horowitz published an article in this Journal featuring the separation of (+)-limonene and L-(−)-carvone from spearmint oil by flash chromatography (14). Herein, we give added detail in the separation and analysis of the spearmint oil components. Our method presents a microscale technique that uses one-fourth as much solvent and silica gel, which is beneficial for programs with large numbers of students. Moreover, added complexity is introduced into this experiment by the separation and presentation of a third component, (1R,2R,4R )dihydrocarveol and by the use of IR spectroscopy as confirmation of the TLC analysis.
tion or be required to discover it on their own by exploring several solvent combinations as part of the learning process. Identification of each component is accomplished through comparison with known standards. The developed TLC plates are visualized first under UV light to identify L-(−)-carvone and then followed by staining with permanganate dip. Exposure to permanganate dip reveals all separated components as yellow–brown spots on a maroon background (Figure 1). Since the background color on the TLC plates gradually flushes brown as permanganate is reduced to manganese dioxide, students should record all of their results before leaving the lab. This initial TLC will serve as a reference in analyzing the collected fractions after the column chromatography of the spearmint oil. Using the procedure described by Reynolds (10), a microscale column is constructed from a Pasteur pipet and loaded with silica gel. The microcolumn is initially saturated with pure hexane until the column appears uniform throughout. When the eluent level becomes even with the layer of silica gel, two drops of spearmint oil are loaded onto the column. Separation of the components is accomplished by increasing polarity of the eluent through a three-stage process,
A
B
Performing the Experiment Students first take a preliminary TLC of spearmint oil to identify the components to be separated and to determine their R f values. We have found that a 10% ethyl acetate兾hexane eluent system provides an ideal separation of the three target components by TLC (Figure 1). At the lab instructor’s discretion, students could be given this informa-
318
Journal of Chemical Education
•
Figure 1. TLC plate of spearmint oil and known standards developed with a 10% ethyl acetate/hexane eluent: (A) the TLC under UV lamp exposure and (B) the same TLC plate after permanganate dip. Samples applied to the different lanes were spearmint oil (s), commercially obtained (+)-limonene (l), L -(−)-carvone (c), and dihydrocarveol (d).
Vol. 84 No. 2 February 2007
•
www.JCE.DivCHED.org
In the Laboratory
beginning with pure hexane and ending with 10% ethyl acetate兾hexane. A total of 15 mL of eluent per student is used to move the three components through the column. Eight fractions (≈2 mL兾fraction) are collected into 10-mm × 100-mm test tubes as the eluent exits the column. When all three stages of the elution process are completed, each of the collected fractions is examined by spotting a small sample from each tube onto a partitioned TLC plate. The partitioned TLC plate is then visualized as described above to determine which fractions contain a component of spearmint oil (Figure 2). A comparative analysis by TLC of all fractions that contained a component of spearmint oil is then performed to verify that purification was successful (Figure 3). If further analysis is desired, pure fractions from three or more students can be pooled in a round bottom flask and concentrated by distillation at reduced pressure in a rotary evaporator. IR spectra of pooled fractions are used to identify the functional groups of each concentrated component. IR spectra of combined fractions containing either (+)-limonene or L-(−)carvone are shown in Figure 4. Furthermore, each experimental spectrum can be analyzed for the major peaks that distinguish the different compounds or compared with the spectra of the commercially purified
Figure 2. Fractions collected by column chromatography spotted directly onto a partitioned TLC plate. The top figure shows the TLC plate under UV lamp exposure and the bottom figure shows the same TLC plate after permanganate dip. Each number of the grid represents a single fraction collected during the chromatography step. The UV lamp exposure (top) shows the fractions containing L(−)-carvone, whereas fractions containing other components are identified after treatment with permanganate dip (bottom).
www.JCE.DivCHED.org
•
A
B
Figure 3. TLC analysis of selected column chromatography fraction developed with a 10% ethyl acetate/hexane eluent: (A) the TLC under UV lamp exposure and (B) the same TLC plate after permanganate dip. Lanes 2–8 represent fractions 2–8 collected during the chromatography step. Positive identification of the different components in the various fractions can be tentatively assigned by comparison with the control plate (see Figure 1).
Figure 4. IR spectra of chromatography fractions containing (+)limonene (A) or L-(−)-carvone (B). Identification of the carbonyl stretch at 1677 cm᎑1 in figure B distinguishes the L-(−)-carvone containing fractions from the those containing (+)-limonene.
Vol. 84 No. 2 February 2007
•
Journal of Chemical Education
319
In the Laboratory
Conclusion The chromatography experiment described herein has successfully been a part of our undergraduate organic laboratory curriculum for the last two years. In a three-hour lab period, students first use thin-layer chromatography to identify an unknown analgesic compound (15), and then as the second part of the lab, perform column chromatography separation of spearmint oil. While students are awaiting development of a TLC plate from the analgesic experiment, they can set up the microcolumn for column chromatography. This experiment teaches students the principles and techniques of column and thin-layer chromatography in an interesting and realistic application. W
Supplemental Material
A detailed student lab procedure, including pre- and post-lab questions, and notes for the instructor are available in this issue of JCE Online. Literature Cited
Figure 5. Comparison of commercial dihydrocarveol (A) with experimentally derived (1R,2R,4R)-dihydrocarveol isolated in fractions 7 and 8 (B). Comparisons of the experimental spectra with the spectra for the known standard samples provide validation in the identification of the different components.
compounds. An example of the comparison between the isolated (1R,2R,4R )-dihydrocarveol and the commercially available dihydrocarveol (diastereomer mix) is shown in Figure 5. Hazards Ethyl acetate, hexane, and spearmint oil are highly flammable. Avoid open flames. Spearmint oil is also an eye and skin irritant. Potassium permanganate is a strong oxidant and will discolor skin and stain clothes.
320
Journal of Chemical Education
•
1. Goodrich, J.; Parker, C.; Phelps, R. J. Chem. Educ. 1993, 70, A158. 2. Svoronos, P.; Sarlo, E. J. Chem. Educ. 1993, 70, A158. 3. Kimbrough, D. R. J. Chem. Educ. 1992, 69, 987. 4. Kandel, M. J. Chem. Educ. 1992, 69, 988. 5. Bird, E. W.; Sturtevant, F. J. Chem. Educ. 1992, 69, 996. 6. Schaber, P. M. J. Chem. Educ. 1985, 62, 1110. 7. Mewaldt, W.; Rodolph, D.; Sady, M. J. Chem. Educ. 1985, 62, 530. 8. Taber, D. F.; Hoerrner, R. S. J. Chem. Educ. 1991, 68, 73. 9. French, L. G. Chem. Educator 2002, 7, 270–277. 10. Reynolds, R. C.; O’Dell, C. A. J. Chem. Educ. 1992, 69, 989. 11. Ruppel, I. B., Jr.; Cuneo, F. L.; Krause, J. G. J. Chem. Educ. 1971, 48, 635–636. 12. Dìaz-Maroto, M. C.; Pérez-Coello, M. S.; Viñas, M. A. G.; Cabezudo, M. D. J. Agric. Food Chem. 2003, 51, 1265– 1269. 13. Dimandja, J. M. D.; Stanfill, S. B.; Grainger, J.; Patterson, D. G., Jr. J. High Resol. Chromatogr. 2000, 23, 208–214. 14. Horowitz, Gail. J. Chem. Educ. 2000, 77, 263–264. 15. Cormier, R. A.; Hudson, W. B.; Siegel, J. A. J. Chem. Educ. 1979, 56, 180.
Vol. 84 No. 2 February 2007
•
www.JCE.DivCHED.org
