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 Abstract 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. Introduction 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 coloration was known as the capsanthin which is xanthophylls abundant in siling labuyo. After the yellow color passed through the column, a red orange color started to form and passed through he column with the use of the second mobile phase solvent which is the combination of hexane and dichloromethane (1:1). The reaction was not that fast as the first one. This red orange color was determined as the anthocyanins and some of the carotenes. After the red orange color, a third mobile phase solvent was used and that is DCM. DCM is more polar compared to hexane. DCM was used to carry the light orange color down the column. Since DCM is polar it carried also polar components of the sample and passed through the column quite slower compared to the second mobile phase reaction.
This light orange color was known as Lutein which is also a naturally occurring carotenoid like carotene. And last after the light orange color was an orange color. The fourth mobile phase solvent which is a combination of Methanol and DCM (1:1) was used to bring the orange color down the column. Since Methanol and DCM is both polar, only methanol is more polar than DCM this solvent carried polar components with them. In comparison with the previous process of carrying components down the column, this fourth mobile phase was the slowest. The orange color was known as carotene which is also an abundant component of siling labuyo.
Then the volume of each was measured using graduated cylinder. In generalizing this experiment,  column chromatography separates substances on the basis of their polarity. The silica gel which is a polar station phase was more polar than any liquid chosen for the mobile phase. Therefore, more polar substances in the sample will adsorb more strongly to the silica stationary phase and elute slowly from the column. In contrast, less polar substances in the sample adsorb weakly to the stationary phase and are rapidly pushed through the column by the mobile phase. Solvent| Color of Fraction| Volume| Hexane| Yellow| 2. 8ml|
Hexane/DCM(1:1)| Red Orange| 1. 5ml| DCM| Light Orange| 4. 3ml| DCM/Methanol(1:1)| Orange| 0. 5ml| Experimental In the extraction of plant pigment, the red pepper was deseeded and chopped into smaller pieces. Then it was triturated with sand and 3ml DCM. After triturating, it was decanted. The decanted plant extract was placed in an evaporating dish with 1/4 anhydrous salt. After drying with anhydrous salt, 10-20 drops of hexane was added. In column packing (micro scale), A small piece of cotton was plugged in a glass dropper. The glass dropper was secured with a burette clamp. Then slurry of silica in hexane was prepared.
After the preparation, a clean dropper was used in placing slurry of silica and quickly packing the micro column to prevent the silica from drying out. Then the solvent on top of the silica was drained. In column loading, a drop of plant extract was placed on top of the silica. This drop was allowed to go down and got adsorbed in the column. In column elution, no liquid extract was on top of the silica. The first eluting solvent (hexane) was added and was allowed to drain down the column. Solvent was added continuously until a color band descends. The color pigment eluted out of the column was collected in a clean, dry, labeled test tube.
The eluting solvent was changed to hexane/DCM (1:1), DCM, and DCM/methanol (1:1). All the pigment samples were collected, covered and protected from direct sunlight. Figure. 1 Extraction Set-up Figure 2. Column Chromatography Set-up References:  Gilbert,J. & Martin S. , “Organic Chemistry Lab Experiments”. 5th edition, Cengage Learning, USA,©2011, pp. 171-185 Retrieved on July 30, 2011 from World Wide Web: http://www. chem. ubc. ca/courseware/121/tutorials/exp3A/columnchrom/ Retrieved on July 30, 2011 from World Wide Web: http://www. mctony. com/columnChromatography. /html