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Column and Thin-Layer Chromatography using the pigments from siling labuyo

R. M. Acedo, A. J. Aguilar, M. A. J. Alcid, K. A. Allan, *J. L. Alvarez

Abstract
Pigments were extracted from siling labuyo with the use of Dicholormethane (DCM)-hexane that acts as eluant. The extract was placed on top of the column using a Pasteur pipette filled with silica gel, eluates were collected using three solvents namely 5 ml DCM-hexane(1:1), 5 ml DCM, 5 ml DCM:MeOH (1:1). The eluates were subjected for chromatography using a Thin Layer Chromatography (TLC) plate. The distance of the pigments were measured and Rf values were determined. (1)

Introduction Methodology Chromatography is a method used to separate and or to analyze complex mixtures. It relies in the differential solubilities or adsorptivities of the components to be separated with respect to two phases, the stationary and the other, mobile. (3) There are various types of Chromatography depending on which two phases are used. In the experiment, Column and Thin Layer Chromatography were used. Column Chromatography is used to separate and purify substantial quantities of components in a mixture for subsequent analysis. In Thin Layer chromatography, the solvent travels up by plate through capillary action. It is usedful for determining the best solvents for a column chromatographic separation.(1) This experiment aims to separate the colored components of siling labuyo using column chromatography, to determine the purity of components using TLC, and measure the Rf values of the colored components in TLC. Pigments of three pieces of siling labuyo were extracted using DCMhexane and eventually pounding it using mortar and pestle with the ratio 1:1. Portions of it were set aside to be use in TLC. Refer to Figure 1.

Figure 1.Extraction of the pigments using DCMhexane

Column was prepared by plugging it with cotton followed by the silica gel packed uniformly up to the indented part of the Pasteur pipette.

Figure 3. The spotting of eluates in the TLC plate.

column

Developing chamber was prepared by placing the approximate amount of DCM-hexane. The developing chamber was covered with watch glass and the TLC plate was allowed to rise up until it reaches the solvent front. Refer to figure 4.

Developing chamber Figure 2. Solvent were introduced in the Pasteur pipette

Figure 2 demonstrates the pigment mixture eluted using 5 ml DCMhexane(1:1), 5 ml DCM, and 5 ml DCM:MeOH (1:1) introduced in portions respectively. The receiver test tubes were changed each time the color of the eluate varies. The number of drops for each color was noted.(2) After collecting the eluates, TLC was performed. The eluates were applied on a 5x8 cm pre-coated TLC plate by equidistantly spotting each color five times. The spot was allowed to dry first before applying the succeeding spots. It was ensured that the spots made were small so that when the plate develops, the colors would not be disarray. Refer to Figure 3.

TLC plate

Figure 4. TLC plate inside the Developing chamber

The components were visualized using UV lamp. Rf values were measured and chromatoplates were documented as seen in Figure 5.

Figure 5. The distance the colored pigments developed.

Results
Table 1. Data obtained from the experiment.

References: Distance traveled Rf in the values TLC plate 3.3 cm 0.57 1.2 cm 1.8 cm 0.21 0.29 1.)http://www.pharmainfo.net/msandhya sravya/blog/column-chromatography 2.)http://www.scribd.com/doc/29411801/ column-and-thin-layer-chromatography 3.)http://www.rpi.edu/dept/chemeng/Biotechenviron/chromo/chromintro.html

Color pigment s Yellow Dark orange Light orange

Eluate (drops) 129 112 94

Discussion Three eluates were yielded from the extraction of the colored components of siling labuyo using column chromatography,yellow, dark orange, and light orange were yielded. The Crude eluate traveled 5.8 cm from the origin: the yellow pigment traveled 3.3 cm: the dark orange traveled 1.2 cm: and the light orange traveled 1.8 cm. Calculation of Rf (Retention factor): The Rf value is the ratio of time spent in the stationary phase relative to time spent in the mobile phase. As seen in Table 1, dark orange pigment has the lowest Rf value which means it is more polar with the stationary phase and the yellow pigment is more polar with the mobile phase. The general formula for computing the Rf value is shown below:

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