Column Chromatography

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Column Chromatography of Plant Pigments

Jaybee Balilea, Sharmaine Baysic, Maria Anjelette Patricia Belen 3Bio-7, Department of Biological Sciences, University of Santo Tomas, Manila, Philippines

Column Chromatography is a form of solid-liquid adsorption chromatography and depends on the essential principles as does in thin layer chromatography. It was used in this experiment in separating and analyzing the different components of Capsicum frutescens (siling labuyo) with the use of solvents such as Hexane (C6H14), Dichloromethane (CH2Cl2), Hexane/Dichloromethane (1:1), and Dichloromethane/Methanol (MeOH) (1:1). The components found in the experiment were carotenoids (carotenes and lutein), xanthophylls (capsanthin) and anthocyanins.


[1]Column Chromatography is a solid-liquid adsorption chromatography where a multicomponent mixture is typically dissolved in a small amount of an appropriate solvent and applied to the top of a packed column containing a finely divided, active solid adsorbent that serves as the stationary phase. An eluant or eluting solvent that serves as the mobile phase is passed down the column. The individual components of a mixture which were adsorbed on the stationary phase at the top of the column begin to move downward with the eluting solvent. These components travel at different rates depending on their relative affinities for the packing material. The more weakly the compound is adsorbed, the faster it will be eluted from the column than a more strongly adsorbed compound. Components are placed in separate test tubes or containers as they exit from the bottom end of the column in bands. Column Chromatography can be used in both analytical and preparative applications. It is used to separate and purity substantial quantities of those components for subsequent analysis.

Column Chromatography was applied in separating the constituent components of Capsicum frutescens. Capsicum frutescens is composed of different constituents such as alkaloids:capsaicin, carotenoids and other pigments, Vitamin A, Vitamin C and other bio-active compounds. It is a rich source of iron and calcium. It is a good anti-fatigue remedy and it helps to boost human’s immune system.

This experiment aims to separate and analyze the different components of Capsicum frutescens with the use of Column Chromatography.

Results and Discussion

In the experiment, Capsicum frutescens was chopped into smaller pieces so that it will be easy to extract its components. These chopped siling labuyo was placed in a mortar and pestle with sand on it. Sand was used to homogenize the extract. Then 3ml dichloromethane (DCM) was added, the desired components of siling labuyo were soluble in DCM. After the addition of DCM, the extract was decanted using funnel into the evaporating dish. After that, an anhydrous salt was added in the plant extract to dry impurities and to promote spontaneous evaporation. After few minutes, Hexane was added in the resulting dry plant extract to liquefy and at the same time to dissolve the sample and the desired components. Then a glass dropper with cotton inside was used as a column. Cotton was inserted to act as a shock absorber. Then a quite dense silica gel was placed in the dropper to serve as the polar stationary phase. Before starting the process or the column elution, the column with silica gel and cotton was rinsed with hexane that produces a slurry texture. After rinsing, the red plant extract was added drop by drop in the column. Then hexane was placed again at the top of the column to act as the first mobile phase solvent. Different solvent was added continuously until a color band descends. Hexane is a non-polar component that interacts with the non-polar component in the sample. This non-polar mobile phase rapidly passed through the stationary phase and eluted faster down the column. As it elutes down the column, the color it carried along was yellow. This yellow...
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