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Column chromatography of photosynthetic plant pigments is a common experiment in introductory laboratory courses for science majors and nonscience majors alike. We report a simple, rapid, reproducible, and small-scale solid−liquid extraction of photosynthetic pigments from inexpensive, storebought dried herbs that affords a dark-green extract solution, all of which can be applied directly to a pipet microcolumn made from as little as 0.75 g of silica gel. Carotenes and chlorophylls are cleanly separated using standard steps in microcolumn chromatography: the former elute together within 20 min using gravity and 9:1 (v/v) heptane:EtOAc, and the latter elute together within seconds using air pressure and EtOAc.
Column chromatography of photosynthetic plant pigments is a common experiment in introductory laboratory courses for science majors and nonscience majors alike. We report a simple, rapid, reproducible, and small-scale solid−liquid extraction of photosynthetic pigments from inexpensive, storebought dried herbs that affords a dark-green extract solution, all of which can be applied directly to a pipet microcolumn made from as little as 0.75 g of silica gel. Carotenes and chlorophylls are cleanly separated using standard steps in microcolumn chromatography: the former elute together within 20 min using gravity and 9:1 (v/v) heptane:EtOAc, and the latter elute together within seconds using air pressure and EtOAc.
JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org.It would seem that a large fraction of the nation's biology students carry out chromatographic separation of leaf pigments, if the frequency of the procedures' inclusion in laboratory manuals and source books is any indication. Although some students perform separations on columns (Dobbins 1972) or thin layers (Foote 1984), it is likely that most classes use paper as the stationary phase. Nearly every published procedure for paper chromatography we have read prescribes extracting leaves with one of several organic solvents, then transferring the extract to the paper. We describe a simpler procedure, one in which the pigments go directly from the leaf to the paper. We have used this method for years, but make no claim to be the first or only ones to employ it. The earliest mention we have found is the brief outline by Knaphus (1967). ProcedureStrips of a good grade of filter paper, such as Whatman #1, should be cut to approximately 15 x 150 mm. Trim one end to a point with scissors, always handling the strip by its edges. Lay the strip on a hard, flat surface. Position a living leaf blade over the filter paper near the trimmed end, then crush a narrow band of leaf tissue into the paper about 2-3 cm above the point. This is accomplished by rolling a small, thick-walled bottle across the leaf while pressing firmly ( Figure 1). Move intact leaf tissue over this band and repeat the procedure a few times to build up a more conspicuous deposit. Try to keep the band as narrow as possible. Without delay, before the deposit has time to dry, hang the strip from a wire hook in a #10 cork and insert this assembly into a 25 x 200 mm test tube. The hook may be fashioned from a paper clip. The bottom of the tube should contain enough developing solution (9 volumes petroleum ether per 1 volume acetone) to immerse the point but not the pigment band. Stand the tube vertically. As the developing solution ascends the paper, several bands of pigment will become evident ( Figure 2).The yellow band at the top contains ,B-carotene and, when some plants are used, a trace amount of a-carotene. The second band from the top is also yellow, but it consists of several xanthophylls, with lutein usually being the most abundant. That the two yellow bands differ in chemical composition may be shown by immersing the upper portion of the fully developed chromatogram in hexane. This action will remove the carotenes, which dissolve in hexane, whereas the insoluble xanthophylls will remain on the paper. Farther down is a bluegreen band of chlorophyll a, followed by the yellow-green band of chlorophyll b. A brownish band will be found at the site of the original deposit when certain kinds of plants are used. Our best gues...
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