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Galica, Maiden M. Garduce, Franzes Rizelle H. Gaspar and Shera Heart M. Go Group 6 2C Medical Technology Organic Chemistry Laboratory ABSTRACT
Chromatography is a way of separating and analyzing the constituents of a mixture by passing them through an inert stationary environment using a mobile solvent that carries them. The two different types of Chromatography used to achieve the objectives of the experiment were the Column Chromatography, which is a preparative technique where the eluates were taken, and the Thin Layer Chromatography, which is a fast technique that required only a small amount of the material. The objectives of this experiment were to illustrate the principles of chromatographic separations, and to demonstrate how Thin Layer Chromatography and Column Chromatography are used in Organic Chemistry. In the experiment, eluates collected from the extract of Malunggay leaves using the process of Column Chromatography were the yellow-colored component, which had 10 drops, and the green-colored component, which had 200 drops; after placing small respective spots of these eluates on the Thin Layer Chromatography plate and placing the plate in a developing chamber for more than five minutes, it was evidently seen that from the origin, the yellow eluate had travelled 3.22 cm and the green eluate had travelled 3.43 cm. The UV lamp was then used to visualize the TLC plate and Rf values were computed, giving the yellow eluate a 0.56 R f value, and the green eluate a 0.60 Rf value.

Chromatography is perhaps the most important technique used to separate the components of a mixture. The separation of mixtures of compounds into the individual components depends on differences in their physical properties and their interaction with the two phases used in Chromatography: the stationary phase, which does not move, and the mobile phase, which moves through the stationary phase. Intermolecular forces between identical and dissimilar molecules, solubility, and vapor pressure are the concepts that must be considered in order to understand Chromatography. Components of a mixture interact with the stationary phase by being adsorbed on it, dissolved in it, or reacted with it. The stationary phase can be a solid, as in Column Chromatography or Thin Layer Chromatography, or a liquid film coated on a solid, as in Paper Chromatography or Gas Chromatography. The mobile phase that flows through the stationary phase is a liquid with the exception of Gas Chromatography where it is a gas, usually helium or nitrogen. If the mobile phase is a liquid, the solubility of each component in the mobile phase is an important factor in determining the rate at which a component moves. The stronger the interaction between a component and the stationary phase, the slower the component is moved by the mobile phase. Separation of components of a mixture using chromatographic techniques depends on differences in their adsorption on the surface of the stationary phase, in their solubility in the stationary and mobile phases, and in their vapor pressures. [1]

Some types of chromatographic techniques are the Thin Layer Chromatography (TLC), Column Chromatography, Gas Chromatography (GC), High Pressure Liquid Chromatography (HPLC), Gel Permeation Chromatography (GPC), and Flash Vacuum Chromatography (FVC). For this experiment, Column Chromatography and Thin Layer Chromatography were the types of Chromatography used. Column Chromatography is one of the most useful methods for the separation and purification of both solids and liquids. This is a solid - liquid technique in which the stationary phase is a solid & mobile phase is a liquid. The principle of column chromatography is based on differential adsorption of substance by the adsorbent. The usual adsorbents employed in column chromatography are silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc., selection of solvent is based on the nature of both the solvent and the adsorbent. The rate at which the components of a mixture are separated depends on the activity of the adsorbent and polarity of the solvent. The adsorbent is placed in a cylindrical tube that is plugged at the bottom by a piece of glass wool or porous disc. The mixture to be separated is dissolved in a suitable solvent and introduced at the top of the column and is allowed to pass through the column. As the mixture moves down through the column, the components are adsorbed at different regions depending on their ability for adsorption, those with lower affinity and adsorption to stationary phase move faster

and are eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last. The different components can be desorbed and collected separately by adding more solvent at the top and this process is known as elution. Elution is best known as the process of dissolving out of the components from the adsorbent, and the solvent is called the eluent. On the other hand, eluate is the solution obtained in the process of elution. [2] Thin Layer Chromatography is a simple, quick, and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture, it is also used to support the identity of a compound in a mixture when the R f of a compound is compared with the R f of a known compound. A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action. As the solvent moves past the spot that was applied, equilibrium is established for each component of the mixture between the molecules of that component, which are adsorbed on the solid, and the molecules, which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. Usually the compounds are not colored, so a UV lamp is used to visualize the plates. [3] The Rf value or the Retention factor is also important in this experiment. The Retention factor is not a physical characteristic of the compound, but is specific to the amount of material spotted, the adsorbent, the solvent, and the temperature. Although exact Rf values cannot be compared on different plates and with different solvents, their relative values provide useful information about the relative polarity of compounds. The Malunggay leaf is the plant used to examine pigments in this experiment. Malunggay leaves have the following pigments: beta

carotone, pheophytin a and b, and chlorophyll a and b. The objectives of this experiment are as follows: (1) To separate colored components of Malunggay leaves using Column Chromatography, (2) to determine the purity of the components using Thin Layer Chromatography, (3) to measure the Rf values of the colored components in Thin Layer Chromatography.

EXPERIMENTAL A. Compounds tested (or Samples used)

Plant used: Malunggay leaves Solvent system used: Hexane:Acetone (1:1)

B. Procedure 1. Extraction Pigments of the Malunggay leaves were first

extracted by triturating together 10 pieces of Malunggay leaves and 10 mL of Acetone:Hexane (1:1) in a mortar and pestle. The extract was divided into two one to be used for the Column Chromatography, and the other, for the Thin Layer Chromatography.

2. Column Chromatography

First, the Column Chromatography set-up was prepared - A Pasteur pipette was plugged with a number of cottons using a wood applicator stick to tamp it down lightly, the column was next uniformly packed with silica gel up to the indented part. The iron clamp connected to the iron stand was used to support the body of the pipette in a vertical position for the entire process of Column Chromatography. Second, 5 mL of the eluents, which are the Hexane:Acetone (1:1), Acetone and Acetone:MeOH, were prepared. Using a Pasteur pipette, Hexane:Acetone (1:1) was first dropped into the column in order to utilize dry packing. As the Hexane:Acetone (1:1) drained and its level had become lower, 0.5 mL of the extract was placed on top of the column using a Pasteur pipette, then the pigment mixture was eluted using another 5 mL of Hexane:Acetone (1:1). Third, drops of eluates were counted and collected in different test tubes for every corresponding color. Colorless eluates were discarded. The column was never allowed to run dry; thus, 5 mL of the second eluent, which is the Acetone, was then dropped in portion into the column in such a way that the column must always contain a volume of liquid higher or equal

to the indented part. Drops of eluates were again counted and collected in different test tubes for every color. 5 mL of the last eluent, which is the Acetone: MeOH was dropped in portion into the column using a Pasteur pipette to elute the pigment mixture. And lastly, drops of its eluates were counted and collected in test tubes varying in their respective colors.

Silica Gel

Figure 2. Thin Layer Chromatography set-up

Figure 1. Column Chromatography set-up 3. Thin Layer Chromatography Using a pencil, a line across the plate about 1 cm from the bottom was lightly drawn. The original extract and the eluates were then applied on the 5 cm x 8 cm precoated TLC plate by spotting 10 times using the colorless end of the capillary tube. Each spot was allowed to dry before applying the next. It was also made sure that the spots were as small as possible. The developing chamber was next prepared by placing an approximate amount of Hexane:Acetone into a beaker; it was made sure that as the TLC plate was placed into the beaker, the solvent system must not reach the spots but rather, it must be lower than the origin. The developing chamber was then covered with a watch glass and was allowed to equilibrate. The solvent system was first allowed to rise up to 1 cm from the upper end before removing the plate from the chamber. After removing the plate from the chamber, the solvent front was immediately marked and the plate was air-dried. The components were then visualized using a UV lamp, the Rf values were measured and the chromatoplates were documented.

Figure 3. Thin Layer Chromatography under UV light


Table 1. Column Chromatography
Color of Components Yellow Green Volume of eluates (Number of drops) 10 drops 200 drops

Using the technique of Column Chromatography, two eluates were yielded from the extraction of the Malunggay leaves. A yellowcolored component and a green-colored component were yielded. The volume of yellow eluate collected was 10 drops; while the volume of the green eluate collected was 200 drops. Also, knowing that the yellow eluate was the first to be eluted out, it can be known that it has lower affinity and adsorption to stationary phase, whereas the green eluate, being the last to be eluted out, has greater adsorption affinity move or travels slower.

Rf values come very handy for identification because one can compare Rf values of the unknown samples with the Rf values of known compounds. Given the results in table 2, it can be seen that there had been no proper separation of components that occurred and this could had been possible due to the following errors that had been committed in the processes of both Column Chromatography and Thin Layer Chromatography: (1) The developing chamber was constantly moved from one place to another, (2) spots were too big, (3) the developing chamber was not covered during the first few minutes, (4) spots were not left completely dried before placing them into the developing chamber. Solving for the Rf value: Distance travelled by the solvent: 5.75 cm Rf value of the yellow component = (Distance travelled by the solute)/(distance travelled by the solvent) = (3.22)/(5.75) = 0.56 Rf value of the green component = (Distance travelled by the solute)/(distance travelled by the solvent) = (3.43)/(5.75) = 0.60 Measuring the distance traveled by the solvent (solvent front) is necessary in order to compute for the Rf value of a component. For the yellowcolored component, given the distance it traveled from the origin of 3.22 and the distance traveled by the solvent, it has an Rf value of 0.56. Whereas the green-colored component, given the distance it traveled from the origin of 3.43 and the distance traveled by the solvent, it has an Rf value of 0.60. The larger an Rf value of a compound, the larger the distance it travels on the TLC plate. When comparing two different compounds run under identical chromatography conditions, the compound with the larger Rf value is less polar because it interacts less strongly with the polar adsorbent on the TLC plate.

Figure 4. Thin Layer Chromatography (result) In the fourth figure, the left-most spot is the yellow eluate and the second spot is the green eluate. From the origin, the yellow eluate had travelled 3.22 cm and the green eluate had travelled 3.43 cm. It was evident that the process of Thin Layer Chromatography was unsuccessful due to the fact that no original extract was spotted beforehand; therefore, no comparison between the distances travelled by the eluates and by the original extract could be made. Also, if viewed under UV light, an unaligned yellow component may be seen at the upper portion of the green eluate, which is also an evident mistake that could have been brought by the disarray of colors because of the often movement of the developing chamber from one place to another. Table 2. Thin Layer Chromatography
Color of Component Yellow Green Distance of component from origin (x) in cm 3.22 3.43 Rf Value

0.56 0.60

After getting the distance travelled by each component in cm, it is necessary to solve for the Rf value of each. Rf value is defined as the distance moved by the solute divided by the distance moved by the solvent. It is necessary to solve for the R f value because a particular compound will travel the same distance along the stationary phase by a specific solvent given that other experimental conditions are kept constant. In other words, every compound has a specific Rf value for every specific solvent and solvent concentration. Also,


In conclusion, the principles of Column Chromatography are somewhat similar to that of the Thin Layer Chromatography. However, one major difference they have is that the Thin Layer Chromatography has a solvent that ascends the plate, whereas in Column Chromatography, the

moving phase travels downward. It had also been concluded that no final judgment of the pigments of the Malunggay leaves could be made in this experiment given that a small part of the procedure was unintentionally skipped.

[1] Pederson, S. and Myers, A. (2011). Understanding the Principles of Organic Chemistry: A Laboratory Course. Canada: Brooks/Cole Cengage Learning. [2] Separation of Compounds Using Column Chromatography. sub=2&brch=191&sim=341&cnt=1 8/2/13 [3] Thin Layer Chromatography. edures/TLC/TLC.html 8/2/13 Bayquen A., et. al. (2009). Laboratory Manual in Organic Chemistry. Quezon City: C & E Publishing , Inc. Pavira, D., Lampman, G., Kriz, G. and Engel, R. (2013). A Microscale Approach to Organic Laboratory Techniques. Canada: Brooks/Cole Cengage Learning. Williamson, K. and Mastors, K. (2012). Techniques Labs for Macroscale & Microscale Organic Experiments. Canada: Brooks/Cole Cengage Learning.