In the Laboratory
Chromatographic Separations Using Solid-Phase Extraction Cartridges: Separation of Wine Phenolics
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Charles A. Brenneman and Susan E. Ebeler* Department of Viticulture and Enology, University of California, Davis, Davis, CA 95616; *
[email protected] Background
Chromatographic Separation by Solid-Phase Extraction Gas chromatography (GC) and high-performance liquid chromatography (HPLC) separations are fundamental to quantitative and qualitative analyses and are used routinely in academic and industrial settings. However, the principles of separation in these systems are often difficult for beginning students to understand owing to the “invisible” nature of the processes occurring on the column and during the analysis. Solid phase extraction (SPE) columns offer a convenient, inexpensive mode of demonstrating chromatographic separations (1). The student serves as the injector, pump (through application of pressure to syringe cartridges or by controlling flow in a vacuum manifold), and detector, since flow of colored analytes through the column can be easily observed. In addition, SPE is a technique that is commonly used in environmental, pharmaceutical, and food laboratories for isolation of organic compounds from a wide variety of matrices. Like traditional liquid–solid column chromatography and HPLC, retention and elution in SPE depend on the interaction of the analyte with the liquid and solid phases. A number of SPE solid phases are commercially available, including normal phase (e.g., silica), reverse phase (e.g., C18, octadecyl), and ion exchange (e.g., benzene sulfonate) sorbents. The polar sites of silica-based stationary phases adsorb polar compounds, and analyte retention and elution are directly related to solvent polarity. Ion-exchange effects, due to the mildly acidic nature of the silica particles, also influence separations on silica-based sorbents. Reaction of the surface silanols of silica with a siloxane derivative results in the formation of bonded silica phases, such as C18 (octadecylsilyl). In general, the nonpolar C18 phases are used to retain nonpolar analytes. Analyte elution in SPE is achieved by selectively desorbing the compound of interest by changing the solvent polarity and/or eluotropic strength. “Strong” solvents match the chemical nature and polarity of the sorbent, whereas “weak” solvents possess characteristics opposite those of the sorbent. Common elution solvents for silica, in order of increasing strength, include hexane, iso-octane, toluene, and dichloromethane. Common solvents for reverse-phase separations, also in order of increasing strength, include water, methanol, isopropyl alcohol, and acetonitrile, and ethyl acetate. SPE sorbents generally utilize relatively large particle and pore sizes (30–60 µm, 60 Å), and analytes are eluted at low pressures. The sorbents are typically packed in syringe-like polyethylene or polypropylene tubes or disks. Detailed reviews of the principles and applications of SPE are available (2–6 ) and several manufactures provide excellent references and other resources for SPE information (7, 8).
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Plant Polyphenols Currently there is much interest in the role of grape and wine consumption in the development of cardiovascular disease and cancer (9–11). Red wine contains high concentrations of a diverse array of polyphenols, a group of natural-product chemicals that have been extensively studied for their biological effects as antioxidants, antimutagens, and cancer preventive agents (12, 13). Polyphenols are also present in a wide range of other fruits and beverages, including blueberries, green tea, and chocolate. Polyphenols contribute to the color as well as to the bitter taste and astringent mouthfeel of these foods (14 ). The Experiment In this experiment, the various classes of phenolics in wine are separated on the basis of differences in polarity. Using reverse-phase (C18) SPE columns and common organic solvents, three fractions are readily obtained: (i) phenolic acids, (ii) catechins (flavan-3-ols) and anthocyanins, and (iii) flavonols (15). The wine pH is adjusted so that acidic polyphenols become ionized, do not absorb to the “neutral” nonpolar C18 column, and are collected in the eluant. After reconditioning the SPE column to acidic conditions, the retained catechins/anthocyanins and flavonols are sequentially eluted by altering the polarity of the eluting solvents, thereby altering the partitioning behavior of the polyphenols between the nonpolar stationary phase and the increasingly nonpolar mobile phase. This separation demonstrates the use of “strong” and “weak” solvents for separation of analytes in a chromatographic separation. Although differences in the color of each fraction are observed, the identity of the major phenolic groups in each fraction can be more accurately determined with a scanning spectrophotometer. Each phenolic class has an absorption band at 280 nm due to the presence of the phenol substituent. In addition, a second band is observed as conjugation is extended from the phenol ring. For example, the phenolic acids have absorption maxima at 320 nm, flavonols at 365 nm, and anthocyanins at 520 nm. These spectra can be used for further discussion and demonstrations of the effect of functional groups and conjugation on spectral properties of molecules. Red wine offers an ideal matrix for this experiment, since several different polyphenol classes are represented. However, in additional experiments, students may be interested in identifying the major phenolic classes in white and blush wines or grape juice. The observed differences represent difference in the amounts and types of polyphenols extracted from grapes during the wine-making process. In general, white and blush wines are made with limited extraction of grape skin and seed polyphenols. This results in low levels of all phenolic
Journal of Chemical Education • Vol. 76 No. 12 December 1999 • JChemEd.chem.wisc.edu
In the Laboratory
classes and the water-soluble phenolic acids predominate. Extraction of catechins, anthocyanins, and flavonols occurs during the production of red wine owing to extended contact with the grape skins and seeds and to the formation of ethanol during fermentation. In summary, this experiment can be used to demonstrate the principles and application of chromatographic separations, using a complex, “real-world” sample. All the materials to perform the experiment are readily obtained. When combined with experiments involving HPLC or GC separations, students gain a greater appreciation for and understanding of the highly automated instrumental systems currently available. In addition, they learn about the chemistry of polyphenolic compounds, a class of natural products that are present in many foods and beverages and are receiving much attention for their potential effects on health. Acknowledgments We thank the students of VEN123 for their contributions to this laboratory experiment and John Ebeler for careful review of this manuscript. Note W A student handout, which includes background information with tables and structures, a materials and equipment list, instructions for carrying out the experiment, and references, is available on JCE Online at http://jchemed.chem.wisc.edu/Journal/issues/1999/Dec/ abs1710.html.
Literature Cited 1. Bidlingmeyer, B. A.; Warren, F. V. Jr. J. Chem. Educ. 1984, 61, 716. 2. Harkey, M. R. In Analytical Aspects of Drug Testing; Deutsch, D. G., Ed.; Wiley: New York, 1989; pp 59–85. 3. Liska, I.; Krupcik, J.; Leclercq, P. A. J. High Resolut. Chromatogr. 1989, 12, 577. 4. Zief, M.; Kiser, R. Am. Lab. 1990, 22, 70. 5. Ebeler, S. E.; Shibamoto, T. In Lipid Chromatographic Analysis; Shibamoto, T., Ed.; Dekker: New York, 1994; pp 1–49. 6. Ebeler, S. E.; Ebeler, J. D. Inform 1996, 7, 1094. 7. McDonald, P. D. Waters Sep-Pak Cartridge Applications Bibliography, 5th ed.; Millipore Corporation: Bedford, MA 1991. 8. Products Catalog and Reference Guide, 1998; J & W Scientific: Folsom, CA, 1998. 9. Bailey, G. S.; Williams, D. E. Food Technol. 1993, 47, 105. 10. Clifford, A. J.; Ebeler, S. E.; Ebeler, J. D.; Bills, N. D.; Hinrichs, S. H.; Teissedre, P.-L.; and Waterhouse, A. L. Am. J. Clin. Nutr. 1996, 64, 748. 11. Watkins, T. R. Wine. Nutritional and Therapeutic Benefits; American Chemical Society: Washington, DC, 1997. 12. Gronbeck, M.; Deis, A.; Sorensen, T. I. A.; Becker, U.; Schnohr, P.; Jensen, G. Br. Med. J. 1995, 310, 1169. 13. Middleton, E.; Kandaswami, C. In The Flavonoids. Advances in Research Since 1986; Harborne, J. B., Ed.; Chapman & Hall: New York, 1994; pp 618–652. 14. Ebeler, S. E. In Functionality of Food Phytochemicals; Johns, T.; Romeo, J. T., Eds.; Plenum: New York, 1997; pp 155–178. 15. Oszmianski, J.; Ramos, T.; Bourzeix, M. Am. J. Enol. Vitic. 1988, 39, 259.
JChemEd.chem.wisc.edu • Vol. 76 No. 12 December 1999 • Journal of Chemical Education
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