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COLUMN AND THIN LAYER CHROMATOGRAPHY

Berba, E.J, Calderon, K.C., Cayabyab, K., Cosare, S.J., *Delos Santos, A.G., Edejer, L.
2D-Pharmacy, Faculty of Pharmacy, University of Santo Tomas

Abstract Chromatography is an analytical procedure used to separate the components of a compound. It aims to identify, purify, analyze, and quantify the mixtures of a specific sample. It is also used to measure the RF value of a constituent. In this experiment, DCM-Hexane is applied to the pigments of Red Chile Pepper or Siling Labuyo to extract its different components. The extracts are introduced to the Column Chromatography (CC) and eluates are collected. The purity of the eluates is then measured using the Thin Layer Chromatography (TLC). With the use of UV Light, the retention or retardation factor of the components from the TLC plate was measured.

Introduction Chromatography is a method of analysis in which the flow of the solvent promotes the separation of the substances by differential movement. In this method, the stationary phase (solid) is placed in a vertical glass column. The mobile phase (liquid) is added to the top and flows down through the column by either gravity or external pressure. A compound that weakly interacts with the stationary phase will spend most of its time in the mobile phase and move rapidly through the column chromatography while the compounds that interact strongly with the stationary phase will move slowly. Column chromatography is generally a purification technique because it isolates desired compounds from a mixture. Thin layer Chromatography is generally used as a qualitative analytic strategy such as checking the purity of the compound or determining the number of components in the mixture. A drop (1-10) of the solution to be separated is placed 1.5 cm form the edge of the plate, and the plate is placed in a container, called a developing chamber, with enough eluting solvent to come to a level just below the point of origin. The solvent moves up through the plate, carrying with it the components of the mixture at different rates. The result, is a series of spots on the plate, falling on a line perpendicular to the solvent level in the container.

The objectives of this experiment are (a) separate the colored components of Siling Labuyo using column chromatography, (b) determine the purity of the components using the thin layer chromatography, and (c) measure the RF values of the colored components. Materials and methods The pigments of the siling labuyo were extracted by cutting it into pieces and by pouring DCM-hexane and eventually triturating it by using a mortar and pestle with the ratio of 1:1. The extracted pigments were set aside for a while. Silica Gel Column was prepared by plugging of the glass column with cotton followed by the silica gel which was uniformly packed and contained no holes until it reached the neck of the Pasteur pipette. 0.5 ml of the extract was placed on top of the column. The pigment mixture was eluted using 10ml DCM-hexane. The solvent was introduced in parts. The column was not allowed to run dry and the colorless eluate collected was discarded. Test tubes were changed each time the color of the eluate changes. The number of drops for each color was counted. After collecting the eluates from the column, Thin Layer Chromatography was performed. The eluates were applied on the TLC plate by equidistantly spotting each spot 10 times. The spot was air-dried before applying the succeeding spots. It was ensured that the spots made were small as possible so that when the plate develops, the colors would not be disarray.

Developing Chamber was prepared by placing the estimated amount of DCM hexane. The inner wall of the chamber was lined with filter paper to let the TLC plate to stand. The chamber was covered with watch glass and was allowed to equilibrate. The TLC plate was carefully introduced in the developing chamber. The solvent system was allowed to rise up until it reaches just 1cm from the upper edge. The TLC plate was then removed carefully from the chamber. The solvent front was immediately marked and the plate was allowed to dry. The components were visualized using the ultraviolet lamp after the plate has developed after elution process which causes substances to appear as colored spots. The RF values were measured and chromatographic plates were documented.

Figure 4. Eluates Collected Results and Discussion Chile Pepper was the specimen used for the experiment on Column and Thin Layer Chromatography. DCM-hexane is the solvent used to extract the different pigments present in the Siling Labuyo. Three eluates were obtained from the extraction of the colored components of siling labuyo using column chromatography. The three different shades of colors were: Red Orange, Light yellow and light orange. The volume of the light yellow eluate collected from the column was 35drops while on the other hand, the volume of the light orange was 85 drops.

Figure 3. Set up for the collecting of eluates

color No. of drops Red Orange Light Yellow Light Orange Table 1. Colors and number of drops each eluate. After collecting the eluates, the group started the preparation for the TLC by spotting the corresponding marked spaces. The original extract is placed at the first marking, followed by the red orange, light yellow and light orange. The TLC chamber was prepared then the Thin Layer Chromatography plate was slowly place inside to allow the process to begin. COLOR OF COMPONENT DISTANCE TRAVELLED BY THE COMPONENT FROM THE ORIGIN RF VALUE

http://www.chemguide.co.uk/analysis/chrom atography/thinlayer.html http://www.chem.ucla.edu/~bacher/General/ 30BL/tips/TLC1.html http://www.tandfonline.com/doi/full/10.1080/ 10826070902956402#.UgujopKw3oI http://www.chemguide.co.uk/analysis/chrom atography/column.html#top

RED ORANGE LIGHT YELLOW LIGHT ORANGE Table 2. thin layer chromatography results

Figure 5. general formula for the RF value

References http://orgchem.colorado.edu/Technique/Pro cedures/Columnchrom/Columnchrom.html http://www.sciencedirect.com/science/article /pii/S0301477008609008 http://courses.chem.psu.edu/chem36/Experi ments/PDF's_for_techniques/CC.pdf http://orgchem.colorado.edu/Technique/Pro cedures/TLC/TLC.html