COLUMN AND THIN LAYER CHROMATOGRAPHY

Alabastro, Reena Avemiel, Albeza, Keeshan Gytha, Arcinas, Ann Iree, Baello, Alexa Jeatrize, Carlos, Chamzelle* Group 1- 2GPH 2013 Faculty of Pharmacy University of Sto. Tomas
ABSTRACT: Chromatography is the most modern and sophisticated method of separating mixtures of substances into their components. This technique is also useful in the identification of compounds. There are various types of chromatography; however the group focused on column and thin layer chromatography. The purpose of the study is to separate the colored components of red siling labuyo using column chromatography, determine the purity of the components using thin layer chromatography and to measure the Rf values of the colored components in thin layer chromatography. In this experiment, the extract of siling labuyo was obtained using DCM-hexane (1:1). The extract was plugged into the column and eluate was collected using column chromatography. In order to determine the purity of the components, eluates were applied to the precoated thin layer chromatography plate. At the end of the experiment developed TLC plate were visualized and the Retention or Retardation Factor was values were measured.

INTRODUCTION Chromatography can be defined as a laboratory technique of separating mixtures of substances into their components. Chromatography is a commonly used method for determining the purity of the components and separating mixtures of liquids into their individual components by using the differential solubilities and adsorptivities of the components to be separated with respect to two phases, one of which is stationary and the other, mobile. 1 The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. 2 A compound that interacts weakly with the stationary phase will spend most of its time in the mobile phase and move rapidly through the chromatographic system. Compounds that interact strongly with the stationary phase will move slowly. This is the underlying principle of chromatography. Familiar example of the application of chromatography includes the separation of colored components of red siling labuyo. 3 There are various types of Chromatography namely solid-liquid (column, thin layer), liquid-liquid (paper, high performance liquid), and gas-liquid (vapour-phase) chromatographic methods. The experiment focused on column and thin layer chromatography. Column chromatography was used to separate the colored components of red siling labuyo extract while thin layer chromatography was then used to determine the purity of the components. The solid may be almost any material that does not dissolve in the liquid phase, and the experiment used silica gel as the solid material. The objectives of the experiment are to (1) separate the colored components of siling labuyo using column chromatography, (2) determine the purity of the components using thin layer chromatography, and (3) to measure the Rf

values of the colored components in thin layer chromatograph. METHODOLOGY A. Materials needed for the experiment The materials used for simple and thin layer chromatography of red siling labuyo extract are: mortar and pestle, Pasteur pipettes, iron stand, iron clamp, beaker, watch glass, capillary tubes, precoated TLC plates, filter paper, siling labuyo, silica gel, cotton, DCM-hexane, DCM and DCM methanol. B. Procedures 1. In the experiment, Column and thin layer chromatography were used. Pigments of the siling labuyo were extracted by cutting it to pieces and by pouring DCMhexane and eventually triturating it by using a mortar and pestle with the ratio of 1:1. 2. Silica Gel Column was prepared by plugging the column with cotton followed by the silica gel which was uniformly packed until it reached the indented part of the Pasteur pipette. 3. 0.5 ml of the extract was placed on top of the column using Pasteur pipette. The pigment mixture was eluted using 10 mL DCM-hexane for red siling labuyo. The system solvent was introduced in portions. 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 varies. The number of drops for each color was noted. 4. After collecting the eluates from the column, Thin Layer Chromatography was performed. The eluates were applied on the5cm X 8cm pre-coated TLC plate by equidistantly spotting each spot 10 times. The spot was allowed to dry first 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. 5. Developing Chamber was prepared by placing the approximate amount of DCM hexane. The inner wall of the chamber was lined with filter paper to allow the TLC plate to stand. The developing chamber was covered with watch glass and was allowed to equilibrate. 6. 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 end. The TLC plate was then removed carefully from the chamber. The solvent front was immediately marked and the plate was allowed to dry.

7. The components were visualized after the plate has developed after elution process and this causes substances to appear as colored spots. The R values were measured and chromatographic plates were documented.

Table 2. Thin Layer Chromatography Color of Components Distance of components from origin (X) in cm Rf value

RESULTS AND DISCUSSIONS Plant used: Red Siling Labuyo Solvent System used: DCM-hexane, DCM and DCM methanol Table 1. Column Chromatography Color of Components Volume of eluates (drops) 1 2 3 4 5 Crude Yellow Orange Red Light Yellow 4.0 5.1 7.2 2.5 0.3 0.5 0.6375 0.9 0.3125 0.0375

The general formula for computing the R value is shown below:

1 2 3 4

Yellow Orange Red Light Yellow

123 110 8 23 After measuring the distance travelled for each spot, the R value (also known as Retardation or Retention Factor was computed) 4 Retardation or Retention Factor is the ratio of time spent in the stationary phase relative to time spent in the mobile phase. Distance of solvent: 8.0 cm

Four eluates were yielded from the extraction of the colored components of red siling labuyo using column chromatography. Four different colors were obtained: Yellow, orange, red and light yellow. The volume of the yellow eluate collected from the column was 123 drops while on the other hand, the volume of the orange was 110 drops, 8 drops from red and 23 drops from light yellow.

Reference to Figure 4, (From right to left) the first spot is the Crude Eluate; the second spot is the first eluate collected from the column and the third spot is the second eluate, fourth spot is the third eluate, and the fifth spot is the fourth eluate collected from the Column Chromatography The crude eluate travelled 4.0 cm from the origin; the yellow eluate travelled 5.1 cm from the origin, the red eluate travelled 7.2 cm, the orange travelled 2.5 cm and the light yellow travelled 0.3 cm. The developed plate wasnt able to show completely the separation of colors. The possible sources of error are from the spotting of the TLC plate. When the extracted pigments of red siling labuyo were spotted on the plate, it was not left completely dry before placing the succeeding spots in addition to that; the spots werent small enough which have caused color the color to disarray. Another source of error is not covering completely the developing chamber during the development of TLC plate. Figure 1. Column Chromatography

Figure 2. TLC plate carefully introduced in the developing chamber

REFERENCES [1] Clark, J. PAPER CHROMATOGRAPHY. http://www.chemguide.co.uk/analysis/chro matography/paper.html (Retrieved last August 03, 2013) [2] Barlao, R.A. COLUMN AND THIN LAYER CHROMATOGRAPHY. http://www.scribd.com/doc/65258518/Pap er-Chromatography-Formal-Report-ORGchem (Retrieved last August 03, 2013) [3] Bayquen, A.V., Cruz, C.T., De Guia, R.M., Lampa,F.F., Pena, G.T., Sarile, A.S. & Torres, P.C. (2009). Laboratory Manual in Organic Chemistry. Manila: C&EPublishing, Inc. p.1314 [4] [No author mentioned]. Thin Layer and Column Chromatography. http://www.xula.edu/chemistry/documents /orgleclab/TLCColProc.pdf (Retrieved last August 03, 2013)

Figure 3. Spotting of Chromatography Plate

Figure 4. Thin Layer Chromatograph