Maggie Xing | mx11 | 10/27/13 | Chem 201-411 | Column Chromatography: Separation of Leaf Pigments

In this lab column chromatography was used to separate the pigments chlorophyll a and b, phenophytin a and b, -carotene, and a xanthophyll from spinach leaves. The pigments were first separated from the spinach leaves by dissolving them in cyclohexane and removing this mixture from the leaves, and then removing the cyclohexane by rotary evaporation. Then the pigments were run through a Pasteur pipette, the column, and were separated into 10 different test tubes according to color. There were seven spots on the TLC plate of the crude spinach extract. The spot with the highest Rf value was large and yellow. It was most likely -carotene, because only carotene and xanthophyll could be yellow, and -carotene is less polar than the other pigments, thus it should travel farther in the TLC plate. The next spot, the grey spot, was likely pheophytin a and b, because it is the only one that can be grey, and it is probably less polar than chlorophyll because it lacks the magnesium ion that chlorophyll has. Chlorophyll B is slightly more polar than chlorophyll A because it has a CHO group instead of a methyl group bonded to the porphyrin. Thus for the blob in the middle which is a lighter green on top and a bluer green on bottom, it is chlorophyll A towards the top and B towards the bottom. Then there are three successive dots that are blue-green, Russian green, and a lighter Russian green. The first is likely chlorophyll B, although it is odd that this chlorophyll B has an Rf value of .35 while the higher one had one of .52. As for the other two, color-wise they should be chlorophyll A, but that doesnt make sense in terms of polarity. They could be other pigments not specified in the lab. Out of all the pigments -carotene is the least polar because it is composed completely of hydrogens and carbons. Hydrogen and carbon have very similar electronegativities, thus their bonds are not very polar, so the molecule cannot be very polar either. The chlorophylls are more polar because they have many substituents

Maggie Xing | mx11 | 10/27/13 | Chem 201-411 | Column Chromatography: Separation of Leaf Pigments

containing nitrogen and oxygen, which have high electronegativities, creating dipole moments. It was initially easy to see the different bands of pigment traveling down the column. First there was an intensely yellow band followed by a lighter green, followed bluegreen one, which makes sense because the yellow dot on the crude TLC plate traveled farthest, thus it should come out first of the column, since the stationary phase in both the column and TLC plate is polar. After the blue-green band, it was more difficult to see the color differences. It became mostly Russian green extract with little variation. Also, it was difficult to spot the corresponding band to the grey dot on the TLC plate. The bands were collected in test tubes based on color, although this became more difficult towards the end because it was difficult to determine if and when the color changed. For the bands that could be discerned, their order matched their order on the TLC plate. We TLC plated four test tubes: the yellow, the blue-green, and the two tubes that we estimated the pheophytins and xanthophyll to be in, respectively. One of the tubes did not show any spots, so it was not included on the Table of Results for the lab. The Rf values on the crude and column TLC plates for the yellow dots were .95 and .94, respectively. The Rf values for the blue-green dots were .52 or .35 and .27, which are not very similar. Similarly, the Rf values for the Russian green dots differed by about .10. Overall, the relative positions of the dots were as expected, but their actual positions showed deviation. If this experiment were to be improved, a better mashing of the initial spinach puree would increase pigment concentration, making them easier to spot. More dots of solution would be loaded onto both TLC plates. Also, while the column was being run, the bands were at an angle so that they were not parallel with the plane of the opening of the column. This may have impacted the visibility of the different band colors.