| Demonstration Experiment on Video Objectives: Chromatographic Method, Plant Pigments Peter Keusch |
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German version
Hazards and safety precautions:
Safety glasses and protective gloves required. The experiment should be performed under a portable fume cupboard giving all-round visibility!
| Extraction of the leaf pigments: Using a pestle, fresh leaves are grinded in a mortar containing 22 mL of acetone, 3 mL of petrol ether and a spatula tip-ful of CaCO3. The pigment extract is filtered. The filtrate is poured into a separation funnel and is mixed with 20 mL of petrol ether and 20 mL of 10% aqueous NaCl solution. The separating funnel is shaken carefully. When the layers have separated the lower layer is allowed to drain into a beaker. This phase is thrown away. The upper layer is washed 3-4 times with 5 mL of dest water. Afterwards the extract is placed in an Erlenmeyer flask and is dried with about 4 spatula tips of Na2SO4. The liquid is carefully decanted into an Erlenmeyer flask. Eluting solvent (mobile phase): mixture of petroleum ether and acetone (7 : 3) Silica gel slurry: Using a beaker of an Appropriate size, a slurry of silica gel and eluting solvent is prepared. Packing of the column: A uniform well-consolidated packing of the column is critical to the success of this chromatographic seperation. A clean, dry column is aligned in a vertical position. A beaker is placed under the column outlet. The column is slowly and evenly filled about two-thirds full with silica gel slurry. The stop cock is opened to allow liquid to drain into the beaker. Pouring the slurry down a glass rod held against the wall of the column will minimize bubbling and turbulence. The side of the chromatographic tube is gently tapped with a cork ring during the packing process, to make the silica gel compact. Meanwhile the stop cock is opened to allow the excess eluting solvent to run out. Using a powder funnel a small amount of sand is carefully added to the top of the silica gel column to prevent it from being disturbed when fresh solvent eluent is added. The solvent level is allowed to drop to 1 mm above sand. The bottom outlet of the separation column is closed. It is very important not to allow the column to run dry. Experimental procedure: Using a volumetric pipet 20 mL of the leaf extract are added directly (or carefully down the side of the column) to the sand layer. Then the mobile phase is drained continuously to the top of the column by aid of a seperation funnel. The bottom outlet of the column is opened. The eluent flows down through the column. The column, with the adsorbent and the sample, is 'developed': As the eluent passes down the column, the components of the mixture begin to move down the column. The separated zones 'flow out' of the column, where the eluates are collected in Erlenmeyers. The flasks are changed as the eluate changes color. Using a swan-neck lamp a bright beam of light is directed at the leaf extract and at the samples eluted from chromatography column. Leaf extract and the samples containing chlorophyll or pheophytin produce a reddish glow. This phenomenon is known as fluorescence. 
 Video clip (Download RealPlayer .rm file)Results and discussion: The mobile phase slowly flows down through the silica gel column by gravity leaving behind zones of color - the chromatogram. The theory of column chromatography is analogous to that of thin-layer chromatography. The different components in the sample mixture pass through the column at different rates due to differences in their partioning behavior between the mobile liquid phase and the stationary phase.
References: Demonstration Experiments on Video
Separation of Food Dyes by Thin Layer Chromatography (TLC)
Demonstration Experiments on Video
Separation of a Lipophilic Dye Mixture by Thin Layer Chromatography (TLC)
Demonstration Experiments on Video
Separation of Plant Pigments by Thin Layer Chromatography (TLC)
Index of Lecture Experiments 
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