Thin Layer Chromatography Lab Report

The purpose of these lab was to help students understand the chromatographic techniques of column chromatography and Thin layer chromatography. Column chromatography is used to help students understand the relationship between eluting power and polarity. Eluting power is defined as the ability of the mobile phase to move a substance from stationary phase. The polarity of the solvent used in the lab can be described as Methanol> acetonitrile>acetone>ethyl acetate> hexane in decreasing order. Since Methanol and acetonitrile are more polar, they will easily separate methylene blue and methyl orange wh while other solvents will take longer time to separate. In the case of hexane, on the dyes did not separate at all and they slightly separated

Furthermore, methylene blue stayed at the top of the column pipette while the methyl orange dye. Since the blue dye stayed at top of the column, one can conclude that it is polar and has greater binding affinity to the stationary phase. The blue dye stayed at the top of the column for almost 4 hours which further helps us conclude that it was a polar dye with greater affinity for the station phase. The eluent collected in the test tube was yellow in color which means that they methyl orange travelled down the column. This also means that methyl orange is less polar and has less binding affinity with the stationary phase. Overall, by using the column chromatography we can conclude that less polar substance will have less binding affinity with stationary phase while polar solvents will bind to the stationary phase for a longer time. This means that polar substance will have longer retention time but lower Rf value while nonpolar substance will have shorter retention time but higher Rf

Two spinach leaves were extracted and grinded using a pestel. The spinach leaves were then mixed with methylene chloride and added to separatory funnel along with water. The organic (green) layer of spinach leaves was then allowed to drain and concentrated in the hood. A melting point capillary was then used to insert some organic solution onto the TLC plates. These TLC plates were then placed in solutions of different ration of acetone and hexane. In this case, the stationary phase was the silica gel plate and the mobile phase was the solvent. According to our observations 4 acetone: 6 hexane and 6 acetone and 40: hexane solutions were able to show the color bands for most spinach pigments. However, the ratio of 4:6 didn’t show the bands for xanthophylls and carotene while 6:4 ratio did show the band for yellow and light yellow which are characteristic of xanthophyll and carotene. We also calculated the Rf factor by measuring how far the substance travelled (ds) and how far the solvent travelled (dx) (Rf=ds/dx). By calculating the Rf values in 6:4 ratio, we can conclude that Xanthophyll (Rf=.84), carotene (Rf=.8), Pheophytin (Rf=.62) were more polar than chlorophyll A (Rf=.89) and Chlorophyll B (Rf=.87) because they had lower calculated Rf