Extraction of Spinach Discussion PDF

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The TA's name was Chathuri Kombala. ...

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Dana Woods Chathuri Kombala Chem 243A 20 September 2015 Extraction of Spinach Discussion For this experiment, the goal was to determine the different pigments that are present in spinach leaves. Since each pigment has a different polarity, we utilized the technique of column chromatography, which separated the different pigments by their polarities. These pigments were then put through thin layer chromatography (TLC) against a crude sample to match up the polarities of the pigments and to confirm that those four pigments were in our sample of spinach. The pigments in spinach are shown to the left. This lab used polarity to separate and identify the pigments. By looking at the known pigments, it is possible to predict the relative polarities of the compounds. Carotene is a hydrocarbon, and does not contain any heteroatoms. This means it is the least polar compound. Pheophytin would be next because it has amine groups, but they are surrounded by carbons and hydrogens. Chlorophyll is third; it has the same structure as pheophytin, but it contains magnesium instead of amine groups, making it slightly more polar. Finally, xanthophyll is the most polar because the

two alcohol groups on the ends allow for hydrogen bonding. After all the tests were completed, our initial predictions were confirmed. The first step was to isolate the different pigments using column chromatography. Chlorophyll a and b, pheophytin a and b, and carotene alpha and beta each separated as only one strand, so there were four main pigments total. With column chromatography, the stationary phase was alumina. Alumina is polar, so the less polar substances will go through the column first while the more polar substances will be attracted to the alumina. For this lab, the pigments were colored, so it was easy to determine when one pigment was finished going through the column. At the end, four test tubes will four different colored pigments were collected. The first test tube was orange (carotene), the second was clear with a green/gray tint (pheophytin), the third one was dark green (chlorophyll), and the fourth was a light green/yellow (xanthophyll). Next, we ran these samples on the TLC plate. The mobile phase chosen was a 75/25 hexane/acetone mixture, which was slightly polar. Silica was used as the stationary phase, which is very polar, allowing us to distinguish the pigments based on how much they moved up the plate. This confirmed our predictions and matched our results from the column chromatography. Carotene was the least polar, with an Rf value of 0.82. Pheophytin was next, with 0.41 being it’s Rf value. The next one was chlorophyll, with an Rf value of 0.21. Lastly, with an Rf value of 0.15, was xanthophyll, confirming that it was the most polar. We also ran a sample of the crude spinach, and four distinct dots showed up. These four dots had similar Rf values to the samples of pure pigments: 0.84, 0.39, 0.22, and 0.11 respectively. The results matched the initial predictions, and upon looking up the pigments, this was the expected outcome of the experiment....