OChem 232 Experiment 15

Rick Politician July 21, 2005 Jesse Hofseth, Luke, Ideed Experiment #15 Isolation of Chlorophyll Pigments from Spinach Purpose: This lab aims to demonstrate to us the potential utility of extraction, column chromatography and thin layer chromatography. It also tries to illustrate that one can separate structurally similar molecules using these precise tools. Data and Results: 0.502 g of spinach weighed Thin Layer Chromatography In the thin layer chromatography experiment the extract and green band spots moved with the edge of the solvent front. Rf values of the extract and green band would therefore be equal to 1. The yellow band spot became invisible to the eye after the TLC plate was placed in the developing jar. Discussion: The extraction process was fairly successful. Our extraction product was a green, but not as dark a green as some of the other groups. This could have been due to a lower concentration of solute in the solution, or lower concentration of chlorophyll in the solute compared to other constituents. The column chromatography created a solid, distinct green band about 2mm in thickness. The hexane eluent washed down a faint yellow carotenoid layer. The green band moved down the column slowly. This could have been due to its strong attraction to the stationary phase (alumina adsorbent). Even if the compounds in the green band were strongly attracted to the alumina, the available bonding sites should have occupied by the acetone, methanol and ether eluents. With no binding sites available, the green band should have followed the rules of gravity and flowed down column with the solvent excess. One other force that could have slowed down the green band was their large size. For instance, molecules like chlorophyll contain almost 50 carbons as well as a long stringy phytyl group. The size and long phytyl would a slow down the chlorophyll in the green band as it tried to squeeze through the packed alumina. The thin layer chromatography failed to separate any of the compounds from each of the spots. Either the stationary phase was too unattractive to all of the compounds, or the mobile phase was way too attractive to all of the compounds. Conclusions: Our extract must have composed of non-polar to slightly polar compounds since all others would have been absorbed into the intermediately polar acetone, or the polar water. The column chromatography showed us that there were two distinct sets of compounds in our extract. The yellow set was more attracted to the slightly polar (70% hexane 30% acetone) solution, while the green set was attracted to some degree to each of the solvents with polarity. Since the stationary phase of the thin layer chromatography experiment did not attract the compounds in the spots, this reaffirmed the fact that all of our compounds are fairly similar in size and polarity. Nevertheless, a more appropriately chosen stationary phase (one slightly more or less polar than the compounds being tested) would likely have separated the spots into their constituent compounds. The experiment showed how a multi-step extraction could isolate a few choice compounds from many other unwanted ones. We learned how a column chromatography allows one to separate compounds based on their relative degree of attraction to the mobile and stationary phases. Lastly we learned that thin layer chromatography is a good method for identifying how many compounds one solution contains, and if those compounds are also contained in other solutions. Questions: 1. Chlorophyll is more polar and thus more attracted to the alumina adsorbent. Also chlorophyll is a larger molecule with a long phytyl carbon chain. Both of these structural issues will slow down the chlorophyll as it bumps into the tightly packed alumina. Chlorophyll would also have a lower Rf value for the reasons. Its polarity would cause it to be more attracted to the stationary phase on the TLC plate. Furthermore, the sized of the molecule would slow the chlorophyll down as it bumps into the stationary phase molecules. Lastly, gravity would have more influence to keep the molecule from rising up the plate. 2. The Rf of the pheophytins is higher than that of chlorophyll, but lower than that of the carotenes. Thus, the pheophytins would have intermediate polarity, intermediate size, or a mixture of the two. We know from the Experiment #15 introduction that the pheophytins are identical to the chlorophylls expect for the central Mg of chlorophylls is replaced by 2 hydrogens. This would imply that pheophytins would be less polar than chlorophyll since the extremely small hydrogens would be inaccessible for bonding, unlike the much larger magnesium molecule. This drop in polarity agrees with the given Rf hierarchy that implies: since the molecules have relatively similar molecular weights, pheophytin should be less polar than chlorophyll 3. The Rf values would increase for the intermediate polar and polar compounds as they would be more attracted to the more polar mobile phase (acetone has intermediate polarity; hexane is non-polar). The non-polar pigments would also be affected by the acetone since the acetone will bind to the available binding sites on the stationary phase, leaving the non-polar compounds with no way to attach to the stationary phase. The non-polar compounds may get caught up in the flow of the solvent and rise faster. Overall, the Rf values of the carotenoids and the chlorophylls would move closer to each other. 4. Since our thin layer chromatography experiment did work out, I cannot use that information to devise an answer. Based on the hierarchy of Rf values presented on page 164 and the Experiment #15 introduction one can make some conclusions. First, the green band may have contained both chlorophylls (green) and pheophytins (gray, would have seen in yellow band, but could be overshadowed in green band). It was mentioned in the lab book that a yellow band may fall out of the green band in the column chromatography. Xanthophylls are both yellow and have a higher polarity than other carotenoids due to their 2 hydroxyl groups. From this information, one might guess that the xanthopylls (green) may also have been contained in the green band.