Thin Layer Chromatography Lab Report
It was found that the concentration of chlorophyll a was 1.590 mol/L, chlorophyll b was 0.9568 mol/L, and this resulted in a total concentration of chlorophyll to be 2.547 mol/L. The concentration of carotenoids was found to be 0.3858 mol/L. When looking at the peaks shown in the absorption spectra it is possible to identify which peak correlates with which compound. Figure 2 contains a peak at around 500 and 700 nm which is a good indicator that that would-be chlorophyll b. This makes sense because this was the bottom band from the thin layer chromatography and chlorophyll b is the most polar compound. Chlorophyll B contains an aldehyde where chlorophyll A has a methyl group making it slightly more polar. Following that, figure 4 contains
a very high peak at 700 nm which is a good indicator that this compound would be chlorophyll A which is slightly less polar resulting in the band being higher up. Lastly, figure 5 is the absorption spectra for the carotenoids because carotenoids are hydrocarbons making them very nonpolar. This would result in this being the very top band with chlorophylls below them because they are polar compounds. The one aspect to take into consideration when looking at these figures is that acetone was used for this laboratory to blank the spectrophotometer. The use of acetone may have been the reason for the absorption spectra not decreasing in absorbance between 500 to 700 nm like it would typically be expected.
Materials and Methods: Five leaves were placed in a mortar with acetone and sand to be ground up. Anhydrous sodium sulfate was added to dry the extract for filtering. Once the sand and remaining leaf bits were filtered out; the absorbance of the mixture was then taken at 661.6, 644.8, and 470 nm in a glass cuvette. A piece of filter paper was then cut into a rectangle with a line 1.5 cm from the bottom where the mixture was applied until there was a dark green band. This filter paper was placed in a chromatography chamber with enough petroleum ether-acetone (90:10) to cover the bottom. Once the solvent rose to 0.5 cm from the top of the paper, it was then removed with a mark made at the solvent line. The portion of each pigment band and the solvent front were measured, and the bands were then cut into pieces. These pieces of each band were set in a test tube with acetone and left for five to ten minutes. Lastly, the absorption spectrum was measured for each fraction from 400 to 700 nm.
a very high peak at 700 nm which is a good indicator that this compound would be chlorophyll A which is slightly less polar resulting in the band being higher up. Lastly, figure 5 is the absorption spectra for the carotenoids because carotenoids are hydrocarbons making them very nonpolar. This would result in this being the very top band with chlorophylls below them because they are polar compounds. The one aspect to take into consideration when looking at these figures is that acetone was used for this laboratory to blank the spectrophotometer. The use of acetone may have been the reason for the absorption spectra not decreasing in absorbance between 500 to 700 nm like it would typically be expected.
Materials and Methods: Five leaves were placed in a mortar with acetone and sand to be ground up. Anhydrous sodium sulfate was added to dry the extract for filtering. Once the sand and remaining leaf bits were filtered out; the absorbance of the mixture was then taken at 661.6, 644.8, and 470 nm in a glass cuvette. A piece of filter paper was then cut into a rectangle with a line 1.5 cm from the bottom where the mixture was applied until there was a dark green band. This filter paper was placed in a chromatography chamber with enough petroleum ether-acetone (90:10) to cover the bottom. Once the solvent rose to 0.5 cm from the top of the paper, it was then removed with a mark made at the solvent line. The portion of each pigment band and the solvent front were measured, and the bands were then cut into pieces. These pieces of each band were set in a test tube with acetone and left for five to ten minutes. Lastly, the absorption spectrum was measured for each fraction from 400 to 700 nm.