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Charles Rollins
Bio-Rad Laboratories Richmond, Caliiornia
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Thin Layer Chromatographic - ~ e ~ a r a t i oofn Leaf Pigments A r a p i d demonstration
Eleven pigment spots can be detected from an extract of grass leaves by a rapid thin layer chromatography (TLC) separat,ionon silicic arid. The technique is simple and makes an interesting demonstration or student experiment. Preparation of pigments. Crush green leaves in a mortar with a few milliliters of alcohol or acetone and twice this volume of petroleum ether. Transfer the solution to a separatory funnel and swirl ~5-ithan equal volume of wat,er. Allow the phases t,o separate and discard the lower water phase. Repeat the water washing twice. Decant the petroleum ether phase t o a bottle and add two grams of anhydrous sodium snlfate. If the pigment solution is too weak, it can be concentrated by e7:aporation with a stream of dry nitrogen. Proced7cre. To 5-g Biosil A-3OB' in a beaker add 12-ml water and stir vigorous1y for 15 sec or nntil thoroughly dispersed. Pour about one milliliter of this mixture onto a clean microscope slide. Spread L T I L Silicic Arid is available from Uio-Rnd Laboratories, 32nd and Griffin. Itichmond, Califomin.
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thc slurry with the stirring rod and tap the slide gently to settle the silicic acid in an even film. 411o\rto set for a couple of minutes then activate by drying in an oven a t l 10°C for 15 mill or longer. When the dried slide has cooled t o room temperature, place one drop of the leaf pigment extract 1.5 cm from one end. Place the slide vertically vith the spot end down in a 250-ml beaker containing a 1-cm layer of henzene:acetone, 7:3 v/v. Cover vith a watch glass. The solvent rapidly rises through the thin layer of silicic acid. Separation of the pigments is apparent almost immediately. Carotenes move the most rapidly followed by chlorophyll a , chlorophyll 6 , and xanthophylls. Eight spots are readily detected and two or t,hree additional spots with more difficulty. Alternatively, the slide may be air-dried overnight. This produces a silicic acid layer of lower adsorptivity. Also, lower percentages of acetone may he used for better separation of chlorophyll a and 17. An alternate procedure is the use of benzene: acetone, 95:s v/v, and continued development for one-half hour d h the cover glass removed from the beaker.
C A R O T E N E S lorongal
Figure 2. Figure 1.
Trocing from TLC separation of green leaf pigments.
32 / Journal of Chemical Education
Photograph of TLC reporation of the same green leaf pigments on different slider.
