Chemical Separation

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Chemical Separation

Introduction:

The objective of this experiment was to extract the pigments from spinach leaves, perform Thin Layer Chromatography (TLC) on the spinach leaf extract, and then determine the best solvent mixture to use to separate the pigments in the extract. The pigments are located inside the chloroplast walls in the cells of the spinach leaves. In order to obtain the pigments the cell walls must be broken down thus exposing the pigment containing chloroplasts. Upon adding a solvent mixture of hexane and acetone, the chlorophyll and carotene pigments can be extracted from the chloroplasts. Further decanting of the dark green fluid from the cloudy light green material will remove impurities from the extracted pigment. To perform thin layer chromatography, a dot of the extracted pigments is placed on a (TLC) plate. By using different ratios of Hexane: Acetone as a mobile phase in (TLC) we should be able to determine which solvent mixture separates the extracted pigments best.

Results:

Ratio (hexane:acetone)
Total Distance (top to bottom)
Color Separations
Rf Values
(9:1)
8.6(cm)
0.8(cm) light green
0.09

7.7(cm) yellow
0.90
(3:2)
9.0(cm)
2.0(cm) yellow
0.22

5.8(cm) yellow/green
0.64

6.0(cm) green
0.67

6.6(cm) light green
0.73
(1:1)
8.4(cm)
3.3(cm) light yellow
0.40

5.8(cm) yellow
0.70

6.5(cm) yellow/green
0.77

6.8(cm) green
0.81

7.1(cm) light green
0.85

8.3(cm) gray
0.99
(2:3)
9.0(cm)
8.0(cm) yellow
0.89

9.0(cm) green
1.00
(1:9)
8.8(cm)
5.2(cm) yellow
0.60

8.3(cm) yellow/green
0.94

*color separations were measured with a ruler in cm

Discussion:

After performing Thin Layer Chromatography on five samples of the extracted spinach pigments, using five different solvent ratios of Hexane: Acetone, a solvent mixture of 1:1 Hexane to Acetone proved to separate the extracted spinach

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