Separating Pigments of Chile Pepper Using Column Chromatography and Thin Layer Chromatography

Freida Coning, Tanya Cruz, Patricia dela Pasion, Joyce De leon, Norina Dimalibot Group 4, 2F Pharmacy Organic Chemistry Laboratory

Chromatography is a method for analyzing complex mixtures by separating them into the chemicals from which they are made. In this experiment, pigments of the Chile Pepper/Cayenne (siling labuyo in Filipino) with the scientific name Capsicum frutescens, were extracted with the use of DCM-hexane(with mole ratio 1:1). Extract was introduced into the column and eluate was collected; this process is the column chromatography method. The purity of the components was determined by using thin layer chromatography. Ultraviolet lamp was used to visualize the developed TLC plate(from the thin layer chromatography) and the Retention Factor was measured for each pigment.

Chromatography is defined as a process used to separate mixtures of substances into their components. They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). There are several types of chromatography. In column chromatography, the stationary phase is a powdered adsorbent which is placed in a vertical glass column. The mixture to be analyzed is loaded on top of this column. The mobile phase is a solvent poured on top of the loaded column. The solvent flows down the column, causing the components of the mixture to distribute between the powdered adsorbent and the solvent, thus (hopefully) separating the components of the mixture so that as the solvent flows out of the bottom of the column, some components elute with early collections and other components elute with late fractions. In thin layer chromatorgraphy, the stationary phase is a powdered adorbent which is fixed to a aluminum, glass, or plastic plate. The mixture to be analyzed is loaded near the bottom of the plate. The plate is placed in a reservoir of solvent so that only the bottom of the plate is submerged. This solvent is the mobile phase; it moves up the plate causing the components of the mixture to distribute between the adsorbent on the

plate and the moving solvent, thus separating the components of the mixture so that the components are separated into separate "spots" appearing from the bottom to the top of the plate.In gas chromatography, the stationary phase is a high-boiling liquid which is packed into a long, narrow glass or metal column. The mixture to be analyzed is loaded by syringe into the beginning of this column. The mobile phase is an inert gas which continuously flows through the column. The components of the mixture distribute between the stationary high-boiling liquid (these components are either condensed or absorbed on the high-boiling liquid) and mobile gas (vapor) phase moving through the column. The gaseous mixture flows through a detector at the end of the column and if it has been successfully separated, the components show as different 'blips' or peaks on a recorder. In chromatography, the retardation factor (also known as retention factor) describes the ratio of time spent in the stationary phase relative to time spent in the mobile phase. In the experiment, the group used column and thin layer chromatography.

The objective of the group¶s experiment is to separate the colored components of the Chile pepper using column chromatography. or elutants. covered and was set aside first.5mL DCM only. another for another 0. It is expressed as Figure 2. A small amount of the mixture to be analyzed is spotted near the bottom of this plate. The pipette is clamped to the iron stand and the test tubes are prepared as receivers.5ml DCM-methanol(1:1) Thin Layer Chromatography(TLC) is a simple. and it slowly rises up the TLC plate by capillary action. This liquid. metal.only the very bottom of the plate is in the liquid. are collected as the solvent drips from the bottom of the column. Using the Pasteur pipette with the cotton in it for bed support. The liquid solvent (the eluent) is passed through the column by gravity or by the application of air pressure. the group filled it with silica up to the indented portion of the glass. R. TLC is also used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound (preferrably both run on the same TLC plate). to prepare the column chromatography set up. and inexpensive procedure that gives the chemist a quick answer as to how many components are in a mixture. they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The Retention factor is computed using this formula: Figure 1. or the eluent. and then eventually the group added 7mL DCMhexane(1:1) was poured. A TLC plate is a sheet of glass. or plastic which is coated with a thin layer of a solid adsorbent (usually silica or alumina). to determine the purity of the pigments using thin layer chromatography and to measure the retardation or retention factor of each component. The mixture is triturated 2-3 times and then the extract from it is collected using a vial. One test tube contains 0. quick. the mixture to be analyzed is applied to the top of the column. Column Chromatography set-up. TLC set-up. The group also prepared the solvents. is the fraction of the sample in the mobile phase at equilibrium.5mL of DCMhexane(1:1). An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Experimental The Chile peppers were triturated using the mortar and pestle until delapitated. is the mobile phase. Because the different components in the mixture have different interactions with the stationary and mobile phases. The retardation factor. In column chromatography. and another test tube for 0. The individual components. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that .

The components were visualized and checked by the UV lamp. 1 2 3 4 COLOR OF COMPONENT Yellow Dark Orange Orange Light Pink VOLUME (number of drops) 100 drops 29 drops 33 drops 23 drops Table 1. Figure 3. It was ensured that the spots are made small so that when the plate develops. The eluates were applied on the TLC plate(5cm x 8cm) by equidistantly spotting each pigment ten times. Thin Layer Chromatography product. Each spot was allowed to dry first before applying the succeeding spots. Thin Layer Chromatography was performed. Column Chromatography set up and the eluates collected. after collecting the eluates. The developing plate was then removed from the chamber carefully. third is the dark orange and last is the light pink. it is eluated by the first solvent. four eluates/colored pigments were yielded. These were the colors yellow. The inner wall was lined by filter paper to allow the TLC plate to stand. orange and light pink respectively. The solvent system was allowed to rise up until it reaches just one centimeter from the upper end. The extract from Chile pepper. For column chromatography. The retention factors were measured after. the group started the Thin Layer Chromatography by spotting each pigment to the TLC plate. The developing chamber was prepared by placing the approximate amount of DCMhexane to a beaker. orange was 33 drops and light pink was 10 drops. Dichloromethane-hexane is used as the solvent system. as we poured the extract and then the eluents(solvents) to the pipette. The extract collected from the six cayennes was three milliliters. As the liquid goes down. putting the extract first on the first spot. The group changes the receiver each time the color varies. the 2mL DCMhexane. Column Chromatography results The volume of the yellow pigment was 100 drops. It is important to equilibrate the . and then covered with watch glass for few minutes for equilibration. With reference to Figure 6. followed by the 2mL DCM and lastly the 2mL DCM-methanol. the developing plate was carefully introduced to the developing chamber. the colors would not disarray. After equilibrating. The number of drops per pigment is also noted. Then the group prepared the TLC chamber and equilibrated it first before placing the TLC plate. Results and Discussion Chile Pepper/Cayennes(siling labuyo in Filipino) was the specimen used for the experiment. dark orange.One milliliter of the extract was introduced to the pipette. second is the yellow pigment. Figure 6. After collecting all the eluates. the dark orange was 29 drops. and the group marked the solvent fronts of each pigment and then air-dried the plate. Figure 4(left) and 5(right).

. E.92.61 Rf value.5.). Also based from table 2. which is the method of extracting one material from another. As the TLC plate was introduced. S. Fedessender R. An Introduction to Chromatography. G. Fedessender. John. Kriz.3cm 1. The solvent travelled up to 6.18 for the orange and zero for the light pink pigment. Experiment and Technology in Organic Chemistry. CA: Academic Press Inc.2cm 0 RETENTION FACTOR 0.92 0. M.. (1994). Haddad. the group immediately measured the distance travelled by the components from the origin to the solvent front.html Retrieved August 11. P. L. the Retardation factors were computed by dividing the distance travelled by the solute or the compound from the distance travelled by the solvent.. Inc. if you divide 6 by 6. Lampman.18 0 Table 2. & Engel. you will get the retention factor of 0.colorado. for the Yellow pigment. (1997). R. Jackson. New York: Chemical Publishing Co. M. 2010 http://orgchem.. I.set up first because this ensures complete distribution of the solvent in the chamber. J. The group learned that one of the processes involved in the experiment was Elution. Thin Layer Chromatography Results After the TLC plate was dried and viewed from the UV lamp to mark the pigments. R. D. & Miller. G. R. G..81 retention factor for the dark orange pigment. (1999).5 cm. Pastro.and the pigments are called the eluates. Hardcourt College Publisher. Pavia. J. Organic Laboratory echniques: A Microscale Approach(3rd Ed. D. S. atography/paper. . 0..chemguide. The distance travelled by the extract was 4cm therefore having a 0.81 0. the group observed as the components went up the plate with the solvent.... J. Therefore. Principles and Practice of Modern Chromatographic Methods.html COLOR OF COMPONENT Yellow Dark Orange Orange Light Pink Distance of the component from the origin in cm 6cm 5. (1998). P.. usually by the means of a solvent. Canada:Brooks. Retrieved August 11. 2010 http://www. References Robards K. TLC/TLC. New Jersey: Prentice The solvent is called the eluent. Same formula was used for the remaining pigments that¶s why the group got 0. & Feist P. The light pink pigment failed to develop a retention factor because the group didn¶t have enough of the eluate for the ten rounds of spotting. (2001) Organic Laboratory Techniques. S. San Diego. T.

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