In the Laboratory
Separation of the Carotenoid Bixin from Annatto Seeds Using Thin-Layer and Column Chromatography James V. McCullagh* and Nicholas Ramos Department of Chemistry and Biochemistry at Manhattan College, Riverdale, NY 10471-4098; *
[email protected] In this experiment, the carotenoid bixin is isolated from annatto (Bixa orellana) seeds using column chromatography. This chromatographic separation has several significant advantages over other plant pigment separation experiments. Significant quantities of the carotenoid can be isolated from small quantities of plant material. Additionally, students are not directly instructed what solvent to use for the separation but instead determine what solvent to use after analyzing several potential solvents by thin-layer chromatography (TLC). The separation of colored plant pigments to demonstrate the principles of TLC and column chromatography has long been a classic experiment in the organic chemistry lab. Typical examples include the separation of chlorophyll from spinach or other plant leaves, the isolation of β-carotene from carrot baby food, and the isolation of lycopene from tomato paste (1–12). This type of experiment is popular because the compounds are brightly colored and students can actually see the separation process as it occurs. These experiments are also more applicationdriven and demonstrate that chromatography can be used to isolate the chemical constituents of items encountered in our daily lives.
Figure 1. Seeds of the annatto or achiote tree (Bixa orellana).
948
Despite their popularity, previous plant pigment separation experiments have several weak points. In previous experiments, the compounds being isolated are found in very small quantities in the plant sources being used and tend to be chemically unstable when isolated (1, 3, 8). Therefore, the separation of these pigments works well as a TLC experiment where the goal is to observe the separation of different pigments, but poorly as a column chromatography experiment where the goal is to isolate significant quantities of the pigments. In many of the literature experiments, the plant pigments are not actually isolated in a pure form at the end of the experiment. Instead the color of the fractions or TLCs are used to see whether the separation worked but the solvent is not removed from the fractions at the end of the experiments to isolate the pure compounds. In the few cases where pigment molecules are isolated, barely detectable quantities are obtained. For example, usually only 0.5 mg of lycopene can be isolated from 3 g of tomato paste (8). In the present experiment, this problem is circumvented because bixin exists in much higher quantities in the plant materials used (typically 4 to 5% by mass) and is significantly more chemically stable than other carotenoids (13, 14). This means that students can isolate significant quantities of the carotenoid from a relatively small quantity of seeds. The other advantage of this experiment is that it demonstrates to students how to choose a solvent for a chromatographic separation. For a practicing organic chemist, being able to determine what solvent to use for the mobile phase is a key skill to master. In this experiment students learn this process by examining the separation ability of five potential solvent mixtures using TLC before determining which one to use for column chromatography. Bixin is isolated in this experiment from the seeds of the annatto or achiote tree (Bixa orellana), a small shrubby evergreen tree native to tropical regions in Central and South America (Figure 1). Historically bixin has been used as a red–orange body dye by the Mayas, Aztecs, Incas, and native Amazonians. It has long been used in these regions to impart color to foods. The dye obtained from this plant was first exported to Europe in the 16th century and its commercial use as a nontoxic naturally occurring dye has been increasing since this time (15–19). Annatto seeds contain several terpenoids and carotenoids that can be readily extracted using typical organic solvents. These compounds include bixin, methyl bixin, norbixin, geranylgeraniol, phytoene, phytofluene, ξ-carotene, and neurosporene among others (Figure 2) (13, 15, 20–23). Of these compounds, bixin is the major carotenoid present in annatto seeds. Even though the compound bixin may not seem familiar, most students will have eaten some of this carotenoid recently. Bixin and its derivatives have been used to produce a yellow to orange color in many types of food products including cheeses (Edam, Munster, Cheshire, etc.), processed cheese foods (Cheez-
Journal of Chemical Education • Vol. 85 No. 7 July 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
In the Laboratory
Whiz and Velveeta), butter, ice cream, meat, fish, cooking oils, margarine, baked goods, and other processed foods (such as cheese puffs) (14, 16, 17, 24). Industrially, bixin is currently the second most used natural color and its use continues to increase as it replaces synthetic food dyes that have been banned from consumer use owing to possible health concerns (18). Beyond use as a food colorant, bixin has been used for other coloring applications. It can be used as an orange dye for natural fibers such as cotton, silk, or wool. Bixin has also been used to color soaps and lacquers (17). Hazards The organic solvents used in this laboratory experiment are volatile, flammable solvents of moderate toxicity but should prove little hazard if handled in a fume hood and disposed of properly after use. Dichloromethane (methylene chloride) is considered toxic and a possible carcinogen. Finely powdered silica gel is irritating to the skin and eyes and can cause lung damage if directly inhaled.
O OR O bixin R = H, Rb = CH3 b methyl bixin R = CH3, R = CH3 norbixin R = H, Rb = H
ORb
OH geranylgeraniol
O O
R
geranylgeraniol esters
phytoene
phytofluene
Discussion and Results This experiment has been successfully used in our firstsemester organic laboratory for the past two years and has been conducted by a total of 97 students to date. The annatto seeds used in this experiment can be purchased in the ethnic foods or spice section of many supermarkets. If they are not available in your location, they can be ordered online from many spice or herb supply companies. A list of several suppliers is included in the online supplement. The experiment is usually conducted with students working in teams of two and the entire isolation procedure can be completed in a single four-hour lab session.1 The isolation of bixin in this experiment is accomplished in three steps. First, the carotenoids are extracted from the annatto seeds with either methlyene chloride or acetone. Second, thin-layer chromatography is used by the students to determine what solvent out of five possible alternatives best separates bixin from the mixture of carotenoids. All of the students successfully chose the correct solvent for the separation after analyzing the TLC plates. Finally, the mixture of carotenoids is separated using column chromatography. Most students isolate between 20 and 25 mg (lowest 11 mg to highest 48 mg) of bixin from 2 g of crushed annatto seeds. The isolated bixin is reasonably pure and shows UV–vis and IR spectra consistent with commercial samples of the compound and matching literature data (22, 23). Conclusion This experiment is an effective means to show students how to conduct a liquid chromatographic separation (TLC and column chromatography; ref 25). This includes the use of thin-layer chromatography to determine an appropriate solvent for a given separation, how to set up and run a chromatography column, and how to analyze the column fractions using thinlayer chromatography. By using brightly colored plant pigments students can directly observe the separation process as it occurs. Students were able to isolate significant quantities of the carotenoid bixin from small quantities of seeds. In addition, UV–vis and IR spectroscopy are a key component of the experiment used to analyze the purity of the isolated product. The isolation of compounds from a plant source that has commercial applications also shows students that chemistry can be a practical and useful field with many applications in our daily lives. This experiment has been used in our organic chemistry lab over the past two years as a replacement to other plant pigment separations experiments. It has worked well with our students and has received favorable feedback. Acknowledgment
Y-carotene
neurosporene
Figure 2. Carotenoids and related compounds found in annatto (Bixa orellana) seeds.
The author would like to thank Suzanne Rudnick for taking the photo used in Figure 1. Note 1. The students can complete up to the end of the isolation of the procedure in a single lab session. The spectroscopy analysis is usually done during free time in the following lab session.
© Division of Chemical Education • www.JCE.DivCHED.org • Vol. 85 No. 7 July 2008 • Journal of Chemical Education
949
In the Laboratory
Literature Cited 1. Williamson, K. L. Microscale Organic Experiments; D. C. Heath and Company: Lexington, MA, 1987; pp 108–118. 2. Wilcox, C. F.; Wilcox, M. F. Experimental Organic Chemistry a Small Scale Approach, 2nd ed.; Prentice Hall Inc.: Englewood Cliffs, NJ, 1995; pp 153–154. 3. Pavia, D. L.; Lampman, G. M.; Kriz, G. S. Introduction to Organic Laboratory Techniques; 3rd ed.; Saunders College Publishing: New York, 1988; pp 286–291. 4. Roberts, R. M.; Gilbert, J. C.; Rodewald, L. B.; Wingrove, A. S. Modern Experimental Organic Chemistry; Saunders College Publishing: New York, 1985; pp 148–149. 5. MacKenzie, C. Experimental Organic Chemistry; Prentice-Hall Inc.: Englewood Cliffs, NJ, 1971; pp 9–18. 6. Palleros, D. R. Experimental Organic Chemistry; John Wiley and Sons Inc.: New York, 2000; pp 190–196. 7. Griffin, G. W.; Quach, H. T.; Steeper, R. L. J. Chem. Educ. 2004, 81, 385–387. 8. Silveira, A.; Evans, J. J. Chem. Educ. 1995, 72, 374–375. 9. McKone, H. J. Chem. Educ. 1979, 56, 676. 10. Kimbrough, D. R. J. Chem. Educ. 1992, 69, 987–988. 11. Mewaldt, W.; Rodolph, D.; Saday, M. J. Chem. Educ. 1985, 62, 530–531. 12. Ronman, P. J. Chem. Educ. 1985, 62, 540. 13. Chemistry and Biochemistry of Plant Pigments; Goodwin, T. W., Ed.; Academic Press Inc.: London, 1965; pp 79, 519, 521, 529. 14. Epp, D. N. The Chemistry of Food Dyes; Terrific Science Press: Middletown, OH, 2000; p 7. 15. Mercadante, A. Z.; Steck, A.; Pfander, H.; Rodriguez-Amaya, D.; Britton, G. Phytochemistry 1996, 41, 1201–1203. 16. Kemsley, J. Chem. Eng. News 2003, 81 (26), 6. 17. The Colour Index, 3rd ed.; Allen, R. L. M., Clark, C. O., Davies, R. R., Lapworth, M., Miles, L. W. C., Skelly, J. K., Stead, E., Wyles, J. M., Wood, W. E., Appel, W. D., Campbell, K. S., Fusser, L., Gonet, F., Habel, O. F., Inkpen, N. A., Loughlin, J. E., Martin, W. R., Paine, G. P., Schlaeppi, F., Swartzwelder, G. M., Sylvester, C. A., Watson, J. G., Wich, E. A., Eds. The Society of Dyers and Colourists: London, 1971; p 3233. 18. Bouvier, F.; Dogobo, O.; Camera, B. Science 2003, 300, 2089– 2091. 19. Brown, D. Encyclopedia of Herbs, 2nd ed.; DK Publishing Inc.: New York, 2005; p 144.
950
20. Mercadante, A. Z.; Steck, A.; Pfander, H. Phytochemistry 1997, 46, 1379–1383. 21. Mercadante, A. Z.; Steck, A.; Pfander, H. Phytochemistry 1999, 52, 135–139. 22. Jondiko, I.; Pattenden G. Phytochemistry 1989, 28, 3159–3162. 23. Karrer, P.; Jucker, E.; Braude, E. A. Carotenoids; Elsevier Publishing Company: Amsetrdam, 1950; pp 256–271. 24. Kalsec Corporation. http://www.kalsec.com/products/annatto_app. cfm (accessed Mar 2008). 25. For additional articles about chromatography see: Bird, E. W.; Sturtevant, F. J. Chem. Educ. 1992, 69, 996–998. Kandel, M. J. Chem. Educ. 1992, 69, 988–989. Svoronos, P.; Edward, S. J. Chem. Educ. 1993, 70, A158. Reynolds, R. C.; O’Dell, C. A. J. Chem. Educ. 1992, 69, 989–990. Danot, M.; Nahmias, S.; Zoller, U. J. Chem. Educ. 1984, 61, 1019–1020. Rubinson, J. F.; Rubinson, K. A. J. Chem. Educ. 1980, 57, 909–910. Davies, D. R.; Johnson, T. M. J. Chem. Educ. 2007, 84, 318–320. Taber, D.; Hoerrner, R. S. J. Chem. Educ. 1991, 68, 73. Wade, L. G. J. Chem. Educ. 1978, 55, 208. Ruppel, I. B.; Cuneo, F. L. J. Chem. Educ. 1971, 48, 635. Marmor, S. J. Chem. Educ. 1965, 42, 272–273.
Supporting JCE Online Material
http://www.jce.divched.org/Journal/Issues/2008/Jul/abs948.html Abstract and keywords Full text (PDF) Links to cited URLs and JCE articles
Color figures
Supplement
Student handouts
Instructor notes including the UV–vis data, separation flowchart, photos of the TLC plates, and IR spectra
Cover This article is featured on the cover of this issue. See p 883 of the table of contents for a detailed description of the cover. JCE Featured Molecules for July 2008 (see p 1008 for details) Structures of some of the molecules discussed in this article are available in fully manipulable Jmol format in the JCE Digital Library at http://www.JCE.DivCHED.org/JCEWWW/Features/ MonthlyMolecules/2008/Jul/.
Journal of Chemical Education • Vol. 85 No. 7 July 2008 • www.JCE.DivCHED.org • © Division of Chemical Education
