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Column Chromatography Lab

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The purpose of the experiment was to extract, purify and identify pigments from spinach leaves (including chlorophyll a, chlorophyll b and beta carotene). The experiment also compared and contrasted two different methods of separation. The first part of the experiment explored column chromatography and the second part explored thin-layer chromatography.

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The three pigments that can be extracted from spinach (chlorophyll a, chlorophyll b and beta carotene) all differ based on their polarity, which arises from functional groups in their structure. The most effective way to separate pigments from spinach is based on their differences in their polarities and this lab explored two chromatographic methods to do so. Chromatography is the technique of separating organic mixtures by allowing them
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In both cases, the stationary phase is silica, a polar compound in which the pigments are sent to travel through. The silica adsorbs each component of the pigment to different extents. Based on dipole-dipole interactions and H-bonding, the most polar pigment will adhere the greatest to the silica and the least polar pigment will adhere to a lesser extent. In column chromatography, the components of the pigment are carried down the stationary phase via non-polar 8:2 pet ether: acetone mixture named a mobile phase. In thin layer chromatography three mobile phases were tested, 7:3 pet ether: acetone (most polar), 8:2 pet ether: acetone and 9:1 pet ether: acetone mixture (least polar). The ratio to pet ether: acetone will not change the order of the components seperated, just the speed of travel of each. The least polar compound will elute faster than the polar compound as a result of the principle “like attracts like”. The difference between the two types of chromatography is the direction that the mobile phase travels. In column chromatography, it travels downwards and in thin layer chromatography it travels upwards. In each case,

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