Spinach Column Chromatography Lab Report

In order to properly separate the molecules from the spinach extract, throughout the column chromatography, we were required to pay close attention to how the bands were flowing through the column. This entailed monitoring the level of the solvent being used to elute the extract and what type of solvent was being used. Beginning the chromatography, we used hexanes because they were the least polar which extracted the least polar molecule from the extract (carotenes). The carotenes did not want to elute initially, so we barely increased the polarity by adding one drop of acetone to a large portion of hexanes. Once this extract was completed, we switched to using a 75/25 hexanes/acetone solution to elute the next least polar molecule(s) which

After checking their structure, this information proved to be accurate because they are just long, conjugated chains with two aromatic rings. The next molecules to appear in the column chromatography (and be the middle of the pack on the TLC plate) were the pheophytin and chlorophyll. Because their molecules from the extract traveled approximately half the distance of the solvent indicates they are fairly polar, but not a great deal. This is true based on their molecular structures where they both have numerous double bonds, rings, and aldehydes. They differ because at the center of their structure, chlorophyll has a magnesium and the pheophytin has hydrogens. Lastly, the most polar molecule was the xanthophyll which received its polarity from the alcohols bonded to its conjugated chain and aromatic

Beginning with hexanes, the first eluent to come out were the carotenes which were the least polar molecules within the extract. Following the carotenes, pheophytin, and chlorophyll eluted from the column due to the 75/25 mixture of hexanes and acetone. This mixture is slightly more polar due to the acetone being added than simply having hexanes which was used to elute the carotenes. Lastly, pure acetone was used as a solvent to elute the remaining molecule, xanthophyll. Xanthophyll is the most polar molecule and only moved with a very polar molecule - acetone. This order (and these compositions) were chosen because if we were to start with a solvent more polar than hexanes, the carotenes, pheophytin, and/or chlorophyll would have all come out together making it difficult to discern between the three during our analysis. If acetone was used as the beginning solvent, all of the molecules would have come out at the same time, making it nearly impossible to tell the difference during TLC or column chromatography. The mobile phase of the column dictates what molecules filter out and when, so it is crucial to pair the solvent polarity with similar extract