In the Laboratory edited by
The Microscale Laboratory
Arden P. Zipp SUNY-Cortland Cortland, NY 13045
A Simplified Method for the Microscale Extraction of Pigments from Spinach Kimberley R. Cousins* and Kathleen M. Pierson Department of Chemistry, California State University, San Bernardino, 5500 University Parkway, San Bernardino, CA 92407
Thin layer chromatographic analysis of the pigments in spinach is a popular undergraduate experiment performed on the macro (1–5) and semimicro (6, 7 ) scales. Sample preparation for chromatography typically requires crushing the spinach leaves with a mortar and pestle; this procedure is tedious, messy, and increases the opportunity for student inhalation of extraction solvents. An alternative blender method for sample preparation (5) requires the use of organic solvents during the initial extraction, as well as manual separation of solvent from the pulp. Subsequent liquid–liquid extraction using a separatory funnel, as in the macroscale methods, often leads to formation of troublesome emulsions. Macroscale methods also require the use of larger volumes of extraction solvents, which creates environmental and safety concerns. The time taken for extraction is also a factor because the pigments of interest fade upon contact with light and air (5). We have refined the process for extracting pigments from spinach by using a commercial vegetable juicer. Our microscale procedure improves on other extraction methods by reducing the time and effort required to prepare the sample and minimizing the volume of solvent used for extraction. In addition, by reducing the extraction time and thus the opportunity for air oxidation of pigments, a chromatogram of increased quality can be obtained. When 60 student pairs used our sample preparation method, only one group failed to isolate enough pigment for TLC analysis. Many students obtained much more pigment than was needed. Overall the procedure was more efficient and less frustrating for the students, and it created less mess than the larger-scale procedure performed previously. As both preparation time and solvent volume are reduced, this method of sample preparation is appropriate for introducing chromatographic analysis in high school or college laboratories.1 *Corresponding author. Email:
[email protected].
Sample Preparation Procedure CAUTION: Methanol, pentane, and hexane (as well as the commonly used elution solvents) are toxic and flammable and should be used in well-ventilated areas only, away from all flames. The evaporation of the pentane/methanol solvent should be performed in a fume hood. The centrifuge should be properly balanced before use, and should not be opened until it has come to a complete stop. Residual nonaqueous solvent should be stored in an appropriate waste container. Using a commercial juice extractor,2 420 g of fresh spinach produces more than 100 mL spinach juice, enough for 25 students. The juice extractor (the same type of “juicer” promoted by television ads) operates by pulverizing the spinach, squeezing virtually all liquids and some small particles into a collecting trough while retaining the bulk of the spinach pulp in a separate compartment. No water or organic solvent is added during the extraction process. The collected dark-green aqueous suspension of plant fiber and extracts, hereafter called spinach juice, is best used immediately. The spinach juice has also been successfully refrigerated for up to one week in opaque bottles with a minimum of head space, without noticeable degradation. The spinach juice suspension must be shaken vigorously immediately before use. To minimize exposure of the isolated pigments to air and light, thin-layer chromatographic plates and developing chambers are prepared and all equipment is gathered before the extraction of the pigments from the spinach juice. A vacuum filtration apparatus is assembled using a sidearm test tube, neoprene vacuum adapter, and a small Buchner funnel. The funnel is lined with filter paper, and a slurry of 1–1.5 g of Celite (SiO2 filter agent) in 3–5 mL of methanol is poured evenly over the filter paper to a depth of 0.5–1 cm. After turning on the vacuum source, 4 mL of spinach juice is poured carefully through the filter without disturbing the Continued on page 1269
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Journal of Chemical Education • Vol. 75 No. 10 October 1998 • JChemEd.chem.wisc.edu
In the Laboratory
Celite. This is followed immediately by 1 mL of methanol and 7 mL of either pentane or hexane.3 The combined filtrate is transferred to a large stoppered centrifuge tube, 1 mL of water is added, and the resulting mixture is shaken vigorously for 1 min. The tube is then spun in a centrifuge for 1– 2 min, after which time a transparent green top layer should be visible.4 The transparent green top layer is transferred using a Pasteur pipet to a clean test tube. The pentane is evaporated using a stream of nitrogen or by holding the tube over a warm sand or water bath, in the hood, until only a few drops of liquid remain. If all of the solvent is accidentally evaporated, two or three drops of pentane may be added redissolve the green residue. Several spots of the sample are applied to a TLC plate, and the chromatogram is developed as described elsewhere.5 Observed spinach pigments include yellow-orange carotenoids, gray pheophytins, green or blue-green chlorophylls, and yellow xanthophylls, often several spots of each color.6 Acknowledgments We wish to thank the reviewers and column editor for many helpful suggestions. Notes 1. Further efficiencies could be achieved by combining our simplified sample preparation method with recently reported methods for rapid TLC analysis using unconventional elution techniques (8). 2. A Moulinex Juice Extractor, Model M 753, was used according to manufacturer’s directions. Other commercial juicers should give similar results.
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3. There are advantages to using either hexane or pentane in this process. Since hexane is less volatile than pentane, hexane would be less likely to undergo accidental evaporation during vacuum filtration, and less solvent would be inhaled by students. On the other hand, pentane requires shorter evaporation time (or less applied heat) during the concentration step, limiting the opportunity for degradation of the unstable pigments. 4. If no transparent green layer is evidenced at this point, it is likely that the pentane evaporated during the vacuum filtration. In this case, 3 mL of pentane should be added to the tube and the shaking and centrifugation steps should be repeated. 5. We use 3 × 8-cm plastic TLC plates coated with silica gel 60, and a dichloromethane/methanol developing solution, as reported elsewhere (1, 2). Alternative solvent systems for chromatogram development reported include petroleum ether/acetone (4) and isooctane/ acetone/diethyl ether (5). 6. For a more complete discussion of the pigments on the final chromatograms, see ref 5.
Literature Cited 1. Hart, H.; Craine, L. E. Laboratory Manual for Organic Chemistry: A Short Course, 8th ed.; Houghton-Mifflin: Boston, 1991; pp 52–53. 2. Pavia, D. L.; Lampman, G. M.; Kriz, G. S. Introduction to Organic Laboratory Techniques, 3rd ed.; Saunders: Fort Worth; pp 286–291. 3. Rollins, C. J. Chem. Educ. 1963, 40, 32–33. 4. Foote, J. Am. Biol. Teach. 1984, 46, 226–9. 5. Strain, H. H.; Sherma, J. J. Chem. Educ. 1969, 46, 476–483. 6. Roberts, R. M.; Gilbert, J. C.; Martin, S.F. Experimental Organic Chemistry: A Miniscale Approach; Saunders: Fort Worth, 1994; pp 157–158. 7. Williamson, K. L.; Heath, D. C. Macroscale and Microscale Organic Experiments, Heath: Lexington, MA, 1989; pp 146–148. 8. Anwar, J.; Nagra, S. A.; Nagi, M. J. Chem. Educ. 1996, 73, 977–979.
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Experiments, laboratory exercises, lecture demonstrations, and other descriptions of the use of chemicals, apparatus, instruments, computers, and computer interfaces are presented in the Journal of Chemical Education as illustrative of new or improved ideas of concepts in chemistry instruction and are directed at qualified teachers. Although every effort is made to assure and encourage safe practices and safe use of chemicals, the Journal of Chemical Education cannot assume responsibility for uses made of its published materials. Many chemicals are hazardous. Precautions for the safe use of hazardous chemicals and directions for their proper disposal are described in the Material Safety Data Sheets and on the labels. We strongly urge all those planning to use materials from our pages to make choices and to develop procedures for laboratory and classroom safety in accordance with local needs and situations.
JChemEd.chem.wisc.edu • Vol. 75 No. 10 October 1998 • Journal of Chemical Education
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