Separation of the Components of ‘Siling Labuyo’ by Column Chromatography and Thin Layer Chromatography

Carmina Soyangco, Martin Sto. Tomas, Michael Tan, Tiffany Jill Te, Ilona Grace Tiburcio, Chloe Sacha Tolentino Group 8, 2-E Medical Technology, Faculty of Pharmacy, University of Santo Tomas


Chromatography is an analytical or separatory procedure based on the adsorption of the components. This experiment aims to separate the different colored pigments of siling labuyo with the use of the DCM-hexane, which served as an extracting solvent, measure the purity of eluates and determine the retention factor of the components. The extracted pigment of siling labuyo was introduced to Column Chromatography and produced two colored pigments or eluates. The eluates collected were light yellow and yellow or light orange. Eluates were used for Thin Layer Chromatography to determine their purities. Retention factor of the components were measured from the developed TLC plate with the use of UV light.

INTRODUCTION Chromatography is defined as a method of analysis in which the flow of solvent promotes the separation of substances by differential migration. It is composed of two phases namely, stationary phase and the mobile phase. Mobile phase is the component which moves the analyte across a stationary phase while the stationary phase prevents the movement of the analyte. Chromatography can be of many types but this experiment focuses on only two types. First is Column Chromatography. It is a method that is based on partition or adsorption and a preparative separation or precise quantitative analysis of nonvolatile organic compounds.
Figure 1: Column Chromatography

Second is Thin Layer Chromatography. It relies on the difference in affinities of compounds like Column Chromatography. It is more often used because of the simplicity of the process

5 milliliters of the extract was first added in the column followed by the three.5 milliliters of the extract was used and added to the column. The eluates Retention factor of each component is different from one another. Colorless eluate was discarded while different colored eluates were separated into test tubes. A solvent that is less strongly adsorbed than the sample is passed through the column and it is known as eluate. Two milliliters of the extracting solvent. The 0.and purity of the components can be measured. UV light was used to visualize the components and to measure the retention factor. Cotton was . making the spots as small as possible. A Pasteur pipette was prepared for column chromatography. Silica gel is a common adsorbent used in column chromatography 0. DCM-hexane was added to get the extract. MATERIAL AND METHODS Materials In the experiment. DCM-hexane. DCM and DCM-MeOH and two milliliters of each were added consecutively. absorbent. It depends on the solvent used. siling labuyo was pounded with mortar and pestle and two milliliters of DCM-hexane was combined to get 0. A developing chamber was also prepared by putting approximate amount of the extracting solvent. DCM-hexane and lined the inner wall with filter paper covered with watch glass. DCM-MeOH respectively. amount of the pigment spotted and the temperature.5 milliliters of extract needed for column chromatography. Method To start the experiment. Three eluents were used. two milliliter-eluents. For thin layer chromatography. DCMhexane. DCM. measure the purity of components and calculate the retention factor of each component by the equation: plugged in the pipette and uniformly packed with silica gel until it reached the intended part of the pipette. the eluates collected were spotted 10 times in a TLC plate. mortar and pestle was used for the grinding of siling labuyo. Few drops of the extracting solvent were added to moisten the silica gel. Figure 2: Thin Layer Chromatography This experiment aims to separate the different colored pigments of siling labuyo.

13 In thin layer chromatography.13 Table 2: Thin Layer Chromatography Table of Results Color of component Light yellow Light Orange (yellow) Volume of eluate (drops) 81 drops 63 drops Color of component Light yellow Distance from point of origin 0 cm 0. its retention factor is 0 while the retention factor of light orange or yellow is 0. RESULTS AND DISCUSSION In column chromatography. two eluates were collected.produced were separated into different test tubes based on the color of the pigment.13. the distance travelled by the components from the origin were measured as well as the retention factor. light yellow and yellow or light orange. The eluates collected were spotted ten times on the TLC plate. The plate was taken out of the chamber after the solvent system was above 1 cm already and left to dry. Table 1: Column Chromatography Table of Results solvent system with a 0.9 cm. 81 drops of the light yellow eluate was collected while 63 drops of the light orange eluate was obtained.9 cm from the origin. Colorless eluate was discarded.9 cm Rf value 0 0. 1 cm above the plate marked by a horizontal line. only the light orange developed in the REFERENCES Light Orange (yellow) . The TLC plate was visualize under UV light. The solvent system travelled at a distance of 6. Since the pale yellow pigment did not develop. The TLC plate was placed in the developing chamber and was allowed to equilibrate. Figure 3: Developed TLC plate Light yellow Light orange Crude Extract Light yellow: Rf = =0 = Light orange/yellow: Rf = 0.

uk/analysis/chromatography/column. USA: Reinhold Publishing Corporation pg. New York.chem. New York and London Academic Press pg. 80-84 Heftmann. from http://www.ucla. (2007). 2nd Edition.wisc. Thin-Layer COLUMN CHROMATOGRAPHY retrieved 2007. K.Books Randerath.chemguide. Chromatography. 11-15 Websites THIN LAYER CHROMATOGRAPHY from http://www.pdf THIN LAYER CHROMATOGRAPHY http://www. (2006).html .

Sign up to vote on this title
UsefulNot useful