ABSTRACT
Column chromatography is a separation process in which the sample is allowed to flow through a column packing with a materials inside the column. It consists of stationary phase which usually a silica gel or alumina and mobile phase such as petroleum ether. In this experiment, the silica gel beads was used as the stationary phase and the petroleum ether as the mobile phase. On the other hand, the sample chosen to be separated was extract of leaf from Mengkudu. Mengkudu. Basically, the process start with the addition of silica gel beads to fill the column until it filled over a half of the column¶s height. Then, the mobile phase was added so as to move the sample inside the column. After that, 1 ml of sample was introduced into the column and the separation of pigments was allowed to occur. Importantly the mobile phase needed to be added at time in which the column has become dried so as not to restrict the movement of the sample. After the process, a clear separation of pigments with different colours was pbserved. Based on the results obtained, 6 pigments was identified that are carotenes, pheophytin, chlorophylls a, chlorophylls b, lutein, xantophylls. INTRODUCT ION
Organic or inorganic compounds, whether obtained by synthesis or from natural sources such as plants or ores, are generally in the form of a mixture. Their separation into individual, pure compounds is essential if they are to be of any use. Chromatography is a physical method of separating a mixture of compounds. A chromatography experiment is carried out in order to know the number of compounds in a sample, identify the compounds, know the relative quantities of the compounds in a sample and to know the effect quantitative separation of the compounds. In chromatography, two phases are necessary to effect the overall separation of the compounds in the mixture analyzed. They are stationary phase and mobile phase. Stationary phase is a phase that does not move. It can be a solid or liquid. On the other hand, mobile phase is a phase that moves and it can be a liquid or gas. In this experiment, we focus on column chromatography which a type of chromatography that used usually for separation and purification of reaction products. An extract of leaf containing several pigments was chosen to be analyzed. In this type of chromatography, finely powdered, porous solid silica gel, which constitute the stationary phase, are packed into a burette-like glass tube, commonly called the column. The column is clamp vertically. The mixture of compounds
to be separated are dissolved in a very small volume of organic solvent and applied as a narrow band at the front end of the column. A liquid which is generally an organic solvent such as nhexane, benzene, chloroform, constituting the mobile phase, is allowed to flow through the stationary phase by gravity. The compounds in the mixture have different adsorption strengths towards the material of the stationary phase. Therefore, the compounds in the mixture, when allowed to move down the column, move with different speeds. Weakly adsorped compounds move faster than the strongly adsorped ones. The different speeds of movement of compounds in the column is known as the differential migration. At the end of the process, the compounds get separated one from another. MATER IALS AND METHOD
In this experiment, the materials involved are silica gel, petroleum ether, sample of leaf extracts (mengkudu), the column, dropper and retort stand. First of all, the colum was packed with the silica gel beads. At the same time, petroleum ether as the mobile phase was added to at certain interval to make sure that the silica gel does not dry. When the addition of silica gel was already reach at the top of the column, then 1ml of leaf extract was added. The sample was allowed to flow through the column and collected as fraction of eluant. Each eluant represent a compound with different colour. Finally, analysis of the fraction was co nducted to identify the compound. RESULTS
Figure 1: Components obtained after separation of leaf extract using column chromatography
DISCUSSION
Column chromatography is often used to purify compounds made in the lab. The materials observed in this experiment is leaf extract of Mengkudu. For column chromatography, the basic principle is actually similar with thin layer chromatograpgy. It consists of mobile phase and stationary phase. For this experiment, the mobile phase is petroleum ether and the stationary phase is silica gel. Based on the result, thereotically a compound which have stronger attraction to mobile phase will eluted first from the column. From the result, the mobile phase which is petroleum ether was eluted first. This is because it is highly nonpolar solvent with chemical formula C6H6Mg Br. The existing benzene ring make it most non polar than the others because it has low dielecrtic constants and are not miscible with water and thus do not attract to the stationary phase that is Silica gel which is polar. Hence, it can be easily eluted from the column. Petroleum ether is a colourless liquid with the odor of gasoline. Structure of petroleum ether is as Figure 2 below :
Figure 2: Chemical structure of petroleum ether On the other hand, the first pigment eluted was carotenes. It has chemical structure as Figure 3 below :
Figure 3: Chemical structure of carotenes
Carotenes is a hydrocarbon with formula C40Hx . It has golden colour.The separation of betacarotene from the mixture of carotenoids is based on the polarity of a compound. Beta-carotene is a non-polar compound, so it is separated with a non-polar solvent such as hexane. In this experiment, is it separated by petroleum ether. A large number of branching methyl group make it less polar then the other pigment. As a result, it form a strong attraction with non polar petroleum ether and easier to be eluted. Apart from that, the second pigment eluted was pheophytin. It has olive green colour. The chemical structure of pheophytin is as Figure 4 below:
Figure 4: Chemical structure of pheophytin Pheophytin has a more polar interaction than carotenes. Pheophytin or Pheo is a chlorophyll molecule lacking a central Mg2+ ion that serves as the first electron carrier intermediate in the electron transfer pathway of photosystem II in plants and the photosynthetic reaction center RC P870 found in purple bacteria. After that, the next molecules eluted was chlorpophylls a. It has blue green in colour. Chlorophyll a is the major pigment involved in trapping light energy and converting it in to electrical and chemical energy. It acts as a reaction centre. structure of chlorophylls a is as Figure 5 below :
Figure 5: Chemical structure of Chlorophyll a
Molecular
It has molecular formula of C55H72O5N4Mg and melting point 117-120°C. The molecular structure of chlorophyll consists of a N-ring with a Mg center, side chains, and a hydrocarbon tail. The fourth pigment eluted was chlorophylls b. it has yellow green colour with molecular formula C55H70O6N4Mg. it replace methyl group in C7 in chlorpohylls a with aldehyde group that make is more polar than chlorophylls a and thus having stronger affinity to silica gel bead and retain longer in the column. Molecular structure of chlorophyll b can be shown in Figure 6 below:
Figure 6: Chemical structure of chlorophylls b Later on, Lutein was eluted. It has yellow colour and has it has almost similar structure to carotenes and xanthophylls. Lutein is present in plants as fatty-acid esters, with one or two fatty acids bound to the two hydroxyl-groups. Chemical structure of lutein was shown as Figure 7 below :
Figure 7: Chemical structure of Lutein Two hydroxyl group in Lutein can interact with hydroxyl group on the surface of silica and make it more difficult to be eluted from the co lumn. Finally, the last pigment eluted was xanto phylls. The chemical structure of xantophylls can be shown in Figure 8 below:
Figure 8: Chemical structure of Xanthophylls Xanthophylls either contain hydroxyl groups and/or pairs of hydrogen atoms that are substituted by oxygen atoms. For this reason they are more polar than the purely hydrocarbon carotenes. As it is more polar than carotenes, it create stronger attraction with Silica gel and hence eluted slower than carotenes. In this experiment, there are several precaution steps need to be followed. One of them is never leave the column in dry condition as it will block the flow of mobile phase to elute the pigment. Top up the column with mobile phase when it almost get lack of the mobile phase. Besides, take serious attention when ha ndling with petroleum ether as it is flammable. CONCLUSION
Column chromatography can be used to separate and purify leaf extract of Mengkudu. Generally, there are seven component can be separated that are the mobile phase, carotene, xanthophylls, lutein, pheophytin, chlorophylls a and chlorophylls b.
REFERENCES
1. Still, W. C.; Kahn, M.; Mitra, A. J. Ogg . chem 1978, 43(14) 2. Fair, J. D.; Kormos, C. M. J. Chromatog r. A 2008, 1211(1-2), 49-54
