Column Chromatography Lab

The purpose of this lab was to extract chlorophyll and carotenoid pigments from fresh spinach leaves and separate and analyze these pigments using column chromatography and thin layer chromatography. Acetone was used as a polar solvent to dissolve the more polar pigments first (Xanthophylls, chlorophylls), while hexane was used as a nonpolar solvent to dissolve the more nonpolar pigments such as the carotenes. In addition to being used as the polar solvent, acetone was used to remove the spinach components that were not pigments such as cellulose which is insoluble. The column chromatography worked by eluting the nonpolar carotene pigments first because the alumina is polar and doesn’t absorb the nonpolar carotene. The polar components such

The chlorophylls showed to have a relatively low Rf value with a range of 0.23-0.5 for chlorophyll a and chlorophyll b. The reason for the chlorophyll’s lesser mobility on the column chromatography and their lower Rf values lies in their structure. The chlorophyll consists of polar components in majority and interacts with the polar alumina in the chamber and is therefore slower to run down the chamber. With the thin layer chromatography, a similar incident occurs as the polar chlorophyll interacts with the polar absorbant in the TLC paper and the polar solvent and therefore it does not climb the TLC paper as fast as a nonpolar solvent would. The carotenes have the opposite occur and travel faster along the column and TLC paper, and therefore have a higher Rf value due to their nonpolar quality. Spots 1A and 1B on the TLC paper were hypothesized to be carotenes due to their high Rf value (0.87) and their yellow-orange color. Spots 3,5, and 12 were hypothesized to be Chlorophyll pigments because of their lower Rf values and the green/ blue-green hue of the spots. Spots 2A and 2B were hypothesized to be Pheophytin because of their distinct gray hue, although further analysis is necessary to determine if they are Pheophytin a or b. The rest of the spots were hypothesized to be various xanthophylls due to their high quantity of spots and the yellow color of the spots. Spot 4 was not distinct enough to propose an identity