Experiment 5: Chromatography Padua, R., Pangan, M., Pineda, M., Puno, J., Quan, J. Group 7, 2G Medical Technology, Faculty of Pharmacy, UST
Abstract Chromatography can be defined as a separation technique based on the selective distribution of chemicals between a stationary phase and a mobile phase. The mixture to be separated is placed as a welldefined band or spot on the stationary phase (a solid or liquid) and the mobile phase (a gas or liquid) is allowed to pass through the system (Bell, Clark & Taber, 2001). Fundamentally, chromatography is a technique whereby the physical separation of different adsorbed materials is accomplished in a single adsorbent (Siggia & Stolten, 1956). In this experiment, malunggay leaves (for odd groups) and red siling labuyo (even groups) were used for the chromatographic method. The pigments were extracted by crushing the malunggay with hexane-acetone. The extract was used in Column chromatography, which then separates it further. After isolating and collecting the compounds, it was spotted in a TLC plate and underwent Thin Layer Chromatography. TLC was used for measuring the Rf value of the components. Based on the result of the experiment, malunggay leaves have four colored components present in it. Some colorless substrates were observed and the Retention factor was measured with the use of a UV lamp. (Bayquen et al., 2009).
Introduction Column chromatography is a preparative technique, wherein compounds are isolated for further studies (Palleros, 2000). During column chromatography, compounds of mixtures are separated by having it pass through a column packed with silica gel. Different solvent systems were used to get the eluate of the mixture. Eluates of malunggay usually consists of three colored components namely Xanthophyll (yellow eluate), Chlorophyll A (dark green eluate), and Chlorophyll B (light green/yellow green eluate). Thin Layer chromatography on the other hand, is a primary tool for rapid qualitative analysis and is used to determine the number of components in a sample (Bell, Clark & Taber, 2001). Retention or Retardation factor is measured with the help of TLC.
Materials and Methods The materials used for Column chromatography are malunggay leaves, mortar
and pestle, Pasteur pipette, iron stand, iron clamp, test tubes, silica gel and hexane-acetone (7:3). For the column chromatography set up, a Pasteur pipette served as the column and was plugged with cotton and then filled with silica gel and assembled with an iron clamp and iron stand. The extract of pigments was obtained by mixing and crushing malunggay leaves with hexane-acetone in a mortar. The extract is then collected in a test tube and 0.5 mL of it is placed on top of the column. Solvent systems (hexaneacetone, acetone, and hexane-methyl) were introduced to the set up in order for the colored components to be separated at a different rate. Colored components are collected in test tubes.
Color of Component 1 2 3 4
Yellow Dark green Light green Yellow green
Volume of eluate (drops) 88 64 65 47
Thin Layer Chromatography Color of Component
For Thin Layer Chromatography, the materials used are capillary tubes, beaker, cotton, watch glass, hexane-acetone, filter paper and a 5 cm x 8 cm precoated TLC plate. The chamber was prepared by placing hexaneacetone in a beaker and lining the inner wall with filter paper. It is then covered with the watch glass and cotton in order for it to equilibrate. The extract and eluates were spotted 10 times on the TLC plate 1.5 cm from the edge. A capillary tube is used for the spotting. After spotting, the TLC plate is then put inside the chamber. Allow the solvent system to rise up to 1 cm from the upper end before removing it. Use a UV lamp to measure the Rf values of the components.
1 2 3 4 5
Distance of component from origin in cm Yellow 5.3 cm Dark green 3.3 cm Light green 3.0 cm Green 2.9 cm Yellow 2.8 cm green
Rf Value
0.87 0.54 0.49 0.48 0.46
The yellow component contains Xanthophyll, which is a non-polar compound. This can be confirmed by the column chromatography. It was the first eluate to come out of the column after using hexane-acetone, a non-polar compound. It follows the concept of like dissolves like. Both chlorophyll a and chlorophyll b contain functional groups that make them polar. Thus they dissolved more when acetone is applied to the column. The yellow component travelled the farthest in the TLC plate due to it being a nonpolar compound. Polar compounds stick more to the TLC plate as to compared to non-polar compounds. As time pass, the yellow component eventually fades, keeping only the green components.
References Results and Discussion Tabular Results Column Chromatography
1. Bayquen, A., Cruz, C., de Guia, R., Campa, F., Peña, G., Sarile, A., & Torres, P. (2009). Laboratory Manual in Organic Chemistry. Quezon City: C&E Publishing Inc.
2. Bell, C. Jr., Clark, A., & Taber, D. (2001). Organic Chemistry Laboratory. USA: Brooks/Cole. 3. Column Chromatography. Retrieved from http://orgchem.colorado.edu/Technique/Proc edures/Columnchrom/Columnchrom.html 4. Column Chromatography Notes. Retrieved from http://chem.rochester.edu/~nvd/chromatogra phynotes.html 5. Palleros, D. (2000).Experimental Organic Chemistry. California: John Wiley & Sons Inc. 6. Siggia, S., & Stolten, H. (1956). An Introduction to Modern Organic Analysis. USA: Interscience Publishers Inc.
