4-ColumnChromSpinachF07

4-ColumnChromSpinachF07 - Column Chromatography Separation...

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Column Chromatography: Separation of Leaf Pigments Reading assignment: Fessenden, R.J., Fessenden, J.S., Feist, P. Organic Laboratory Techniques , 3 rd ed.; Brooks/Cole: Pacific Grove, 2001, pgs 49-61 (review of extraction), 119-129 (column chromatography – pay close attention to section 11.3). Introduction Column Chromatography In the previous experiment we used the analytical method of thin-layer chromatography to determine the purity and identity of the isolated samples of caffeine and aspirin. Column chromatography is another example of a separation technique used often to isolate and purify compounds from natural product mixtures (as in today’s lab) or from a reaction mixture. Chances are great that if you looked in the workspace of the organic graduate students upstairs more than 50% would have a “column” going at this very moment! Like thin layer chromatography, column chromatography requires both a mobile and stationary phase. The polar stationary phase (most commonly silica gel, SiO 2 , or alumina, Al 2 O 3 ) is packed into a tall glass cylinder along with the solvent (the mobile phase). The sample, dissolved in a minimum amount of solvent, is loaded (carefully) to the top of the column. The solvent is allowed to flow through the column separating the compounds in the mixture based on their affinity for the solvent versus the adsorbent. When milligram amounts of material are being used, the column can be as small as a Pasteur pipette. This method is especially effective when the fractions are colored because the different fractions are easily identified based solely on visual inspection. More commonly the compounds to be separated are colorless, in which case small fractions are collected and analyzed by TLC. Photosynthesis and Plant Pigments Spinach, like other green plants, contains pigments that make the process of photosynthesis possible. The structures of several spinach pigments are shown on the next page (figure 1). As you will learn in Chem 152, molecules with a large number of consecutive (conjugated) double bonds tend to absorb light in the visible region of the electromagnetic spectrum. The light absorbed by the pigments is converted into chemical energy. Our eyes see the light that is transmitted . As a result highly conjugated molecules, like the pigments of spinach, are colored. (See 15.2C in Loudon for a complete description.)
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Figure 1. The structures of several pigments in spinach: chlorophyll a (blue-green), chlorophyll b (green), pheophytin a and b (grey), carotene (yellow orange) and xanthophyll (yellow).
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