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

ABSTRACT

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

and purity of the components can be measured.

Figure 2: Thin Layer Chromatography

This experiment aims to separate the different colored pigments of siling labuyo, 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. Silica gel is a common adsorbent used in column chromatography 0.5 milliliters of the extract was used and added to the column. Three eluents were used; DCMhexane, DCM and DCM-MeOH and two milliliters of each were added consecutively. A solvent that is less strongly adsorbed than the sample is passed through the column and it is known as eluate. Colorless eluate was discarded while different colored eluates were separated into test tubes. For thin layer chromatography, the eluates collected were spotted 10 times in a TLC plate, making the spots as small as possible. 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. UV light was used to visualize the components and to measure the retention factor. Method To start the experiment, siling labuyo was pounded with mortar and pestle and two milliliters of DCM-hexane was combined to get 0.5 milliliters of extract needed for column chromatography. Few drops of the extracting solvent were added to moisten the silica gel. The 0.5 milliliters of the extract was first added in the column followed by the three, two milliliter-eluents; DCM-hexane, DCM, DCM-MeOH respectively. The eluates

Retention factor of each component is different from one another. It depends on the solvent used, absorbent, amount of the pigment spotted and the temperature. MATERIAL AND METHODS Materials In the experiment, mortar and pestle was used for the grinding of siling labuyo. Two milliliters of the extracting solvent; DCM-hexane was added to get the extract. A Pasteur pipette was prepared for column chromatography. Cotton was

produced were separated into different test tubes based on the color of the pigment. Colorless eluate was discarded. The eluates collected were spotted ten times on the TLC plate, 1 cm above the plate marked by a horizontal line. The TLC plate was placed in the developing chamber and was allowed to equilibrate. The plate was taken out of the chamber after the solvent system was above 1 cm already and left to dry. The TLC plate was visualize under UV light, the distance travelled by the components from the origin were measured as well as the retention factor. RESULTS AND DISCUSSION In column chromatography, two eluates were collected, light yellow and yellow or light orange. 81 drops of the light yellow eluate was collected while 63 drops of the light orange eluate was obtained.
Table 1: Column Chromatography Table of Results

solvent system with a 0.9 cm from the origin. The solvent system travelled at a distance of 6.9 cm. Since the pale yellow pigment did not develop, its retention factor is 0 while the retention factor of light orange or yellow is 0.13.
Figure 3: Developed TLC plate

Light yellow

Light orange

Crude Extract

Light yellow:

Rf =

=0 =

Light orange/yellow:

Rf =

0.13
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 0.9 cm

Rf value

0 0.13

In thin layer chromatography, only the light orange developed in the REFERENCES

Light Orange (yellow)

Books Randerath, K. (2006). Thin-Layer Chromatography. New York and London Academic Press pg. 80-84 Heftmann, E. (2007). Chromatography, 2nd Edition. New York, USA: Reinhold Publishing Corporation pg. 11-15 Websites THIN LAYER CHROMATOGRAPHY from http://www.chem.wisc.edu/courses/342/Fall2004/TLC.pdf THIN LAYER CHROMATOGRAPHY http://www.chem.ucla.edu/~bacher/General/30BL/tips/TLC1.html COLUMN CHROMATOGRAPHY retrieved 2007, from http://www.chemguide.co.uk/analysis/chromatography/column.html