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COLUMN AND THIN LAYER CHROMATOGRAPHY

Alava, P.J., Alcausin, D.A., Andal M.I., Bagon, N.E., *Barretto, D., Bautista, C. 2D-Pharmacy, Faculty of Pharmacy, University of Santo Tomas

Abstract Chromatography is a method used to separate mixtures of substances into their components. The type of chromatography to be employed depends on the two phases used; it may be solid-liquid (column, thin layer), liquid-liquid (paper, high performance liquid), and gasliquid (vapor-phase). In this experiment, the type of chromatographic method used was solidliquid, specifically column chromatography, which is used as the preparative technique, and thin layer chromatography, which is used as the analytical technique. In column chromatography (CC), the components of the malunggay leaves were extracted using the solvent system hexane-acetone and they were introduced into the column, which was filled with cotton and silica gel with which the colored eluates were collected. Using the thin layer chromatography (TLC), the purity of the colored components (green, yellow, green-yellow and moss green) was determined. The Retention factor (Rf) was computed with the result of the yellow component having the highest Rf value (0.886) and the green-yellow component together with one of the Rf values of the moss green component having the lowest Rf value (0.286). Introduction Nowadays, almost all elements and compounds are found in their impure state. Most of the time, they need to be rid of impurities to be utilized in the industry. This necessitates the use of separation and purification processes. One of the most common and widely used separation and purification methods is chromatography. Chromatography is a technique used to separate mixtures of substances into their components, which depends on the different partition coefficient of the components to be separated with respect to the two phases to be used. The mobile phase is a mixture, which moves through the stationary phase, which holds another material. The different types of chromatography depend on the two phases to be used. It may be solid-liquid (column, thin layer), liquid-liquid (paper, high performance liquid), and gas-liquid (vapor-phase). In this experiment, the type of chromatographic method used was solidliquid, specifically column chromatography and thin layer chromatography. Column chromatography (CC) is used as a preparative technique. It separates and purifies individual chemical compounds from mixtures. Often, silica gel or alumina packed in a vertical glass column is used as the stationary phase. The mobile phase is usually a pure solvent or a mixture of pure solvents. Thin layer chromatography (TLC) is used as an analytical technique. It is used in separating non-volatile mixtures, identifying compounds in a given mixture, and/or determining the purity of a substance. The stationary phase is usually a thin coating of silica gel or alumina in sheet of glass, plastic or foil. The mobile phase meanwhile is a solvent or a solvent mixture. The Retention factor (Rf) is the distance travelled by the compound over the distance travelled by the solvent. The larger the Rf of a compound, the larger the distance traveled by the compound on the TLC plate, the less polar it is. The objectives of this experiment are to (a) separate the colored components of malunggay leaves using column chromatography, (b) determine the purity of the components using thin layer chromatography (TLC), and (c) measure the Rf values of the colored components in TLC.

Materials and Methods The materials needed for the extraction of the compounds are malunggay leaves (Moringa oleifera), hexane, acetone and mortar and pestle. The malunggay leaves were crushed on a mortar using a pestle. Adding 1 ml of hexane-acetone (7:3), the extract was obtained, was placed on a test tube and was set aside. Meanwhile, the materials needed for the set-up of the column chromatography are Pasteur pipettes, iron stand, iron clamp, vials, silica gel and cotton. A Pasteur pipette was filled with cotton and uniformly stuffed with silica gel up to its indented part. The Pasteur pipette was set on an iron stand using an iron clamp. For the separation of individual compounds from the extract of malunggay leaves, half an ml of the extract was introduced to the column via a Pasteur pipette. 10 ml of the solvent system hexaneacetone (7:3) was instilled in portions (not letting the column run dry) for elution. The colored eluates were collected separately into different vials according to their color while the colorless eluates were discarded. Simultaneously, the number of drops of each color of the eluates was recorded. The collected eluates along with the crude extract were applied on a 5x8 cm precoated (with alumina/silica gel) thin layer chromatography plate by spotting each color 10 times on the previously drawn 1 inch from the lower end using a capillary tube, allowing each spot to dry before applying the next spot. All the distances of each color's spots should be equal with one another. The materials needed for the set-up of the developing chamber for the thin layer chromatography are 400-ml beaker, watch glass, hexane, acetone and filter paper. The beaker was filled with approximate amount of hexane-acetone (7:3) such that the height of the solvent system will not exceed 1.5 cm. Filter paper was used to cover the inner

wall of the beaker. The set-up was then covered with a watch glass and allowed to equilibrate for the prevention of evaporation of more volatile components ahead of the less volatile components. The precoated TLC plates were placed in the TLC developing chamber and the solvent system was permitted to rise. Once it reached the predrawn line of 1 cm from the upper end, the TLC plates were removed from the TLC developing chamber. The solvent front and the compound fronts were marked and air-dried. The TLC plates were observed under UV light and invisible spots were marked. The distance travelled by the components and the solvent were recorded for the computation of the Rf values. Results and Discussion Using the solvent system hexaneacetone (7:3), four colored eluates were obtained from the process of column chromatography namely Green, Yellow, Green-Yellow and Yellow-Green. Table 1 Column Chromatography
Color of Component Green Yellow Green-Yellow Yellow-Green Volume of Eluate (drops) 21 16 18 18

1 2 3 4

As shown on Table 1, the green eluate produced 21 drops, the highest of the four eluates. The yellow eluate produced 16 drops, the lowest of the eluates, while both the green-yellow and yellow-green eluates produced 18 drops. Thin layer chromatography yielded four different colors from the four colored eluates produced from previously done column chromatography namely green, yellow, green-yellow and moss green.

Having obtained more than one colored component, the malunggay leaves are determined to be an impure substance. Table 2 Thin Layer Chromatography
Color of Component 1 2 3 4 Green Yellow Green-Yellow Moss Green Distance of Component from origin (X) in cm 2.9 2.2 1.9 6.2 2.0 3.3 2.3 2.1 Rf value 0.414 0.314 0.271 0.886 0.286 0.471 0.324 0.286

decreasing distance component.

travelled

by

the

The larger the Rf of a compound, the larger the distance traveled by the compound on the TLC plate, the less polar it is. Applying this statement to the results of the experiment, it can be determined that the least polar of the components is yellow. The most polar, meanwhile, of the components is green-yellow. References Retrieved August 14 2013, from http://www.xula.edu/chemistry/documents/or gleclab/12TLCCol.pdf Retrieved August 14 2013, from http://orgchem.colorado.edu/Technique/Pro cedures/TLC/TLC.html Retrieved August 14 2013, from http://www.chemguide.co.uk/analysis/chrom atography/column.html Retrieved August 14 2013, from http://www.marzkreations.com/Chemistry/Chromatography/1 63a-Chromatography.html Retrieved August 14 2013, from http://anchem.univie.ac.at/fileadmin/user_up load/anchem/Introduction_to_Chromatograp hy.pdf

Each distance of the spot/s of each color from the base line (the 1.5 cm line above the lower end of the plate) was recorded as shown in Table 2. The highest spot, also the one that had the largest distance travelled was obtained from the yellow component, which was 6.2 cm. There were three green components, thus three distances obtained: 2.9, 2.2 and 1.9. Moss green component also had 3 distances: 3.3, 2.3 and 2.1. The lowest spot, also the one that had the shortest distance travelled was the green-yellow component, which was 2.0. The Rf value for each colored component were calculated using the general formula:

With the distance travelled by the solvent as 7 cm, each distance was substituted in the formula producing the Rf/s for each color. Green had 0.414, 0.314 and 0.271 with respect to decreasing distance travelled by the component. Yellow had the highest Rf with 0.886. Green-Yellow had the lowest Rf with 0.286. Moss Green had 0.471, 0.324 and 0.286 with respect to