Column and Thin Layer Chromatography

=========== ORGANIC CHEMISTRY ============

Organic Chemistry Laboratory - CH 201 (2010-2011) Experiment 4B

Column and Thin-Layer Chromatography of Capsicum Frutescens L. Pigments

John Cyril Abanto*, Vernalyn Abarintos and Clarice Gail Abella
Department of Chemistry, College of Science
University of Santo Tomas, Espana Street, Manila 1050

Date Submitted:
September, 2010
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Abstract: The experiment was done to separate and analyze the components of chili pepper. Chromatography was used because of its powerful technique in separating mixtures. In this experiment the Chili pepper pigments was extracted using DCM, the extract was then introduced into the column and eluate was collected, a technique called column Chromatography. Using the Thin Layer Chromatography the purity of the components was tested. The UV lamp was used to spot the UV visible components and the Retention Factor was computed.

Keywords: Column Chromatography, Thin-Layer Chromatography, Retention Factor ____________________________________________________________

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1. Introduction

Chromatography (color writing) is a separation technique used to identify the components of a mixture and to purify a compound. The mixture dissolves in a solvent, the mobile phase, as it passes through an adsorbent material, the stationary phase. Mikhail Tswett developed this process to separate the pigments in green leaves. He dissolved the leaf extract in an organic solvent and let the solution run down through a vertical glass tube with chalk powder. A variety of pigments flowed through the column at different rates, while a series of bands appeared on the white chalk column

Several chromatographic techniques are being used today such as gas chromatography (GC), high-pressure liquid chromatography (HPLC), and thin layer chromatography (TLC). Liquid chromatography, or column chromatography, is patterned from Tswett’s experiment. Solvents are added to into a stationary phase, alumina or silica gel, packed into a glass column. There are two types of column chromatography depending on the flow of solvent: gravity column chromatography and flash chromatography. In flash chromatography, the solvent is forced down the column by positive air pressure; while in gravity column chromatography, the solvent is allowed to flow by gravity, or perlocation.

The objective of this experiment is to separate and analyze pigments of capsicum frutescens, specifically chlorophylls (chlorophylls a and b, pheophytin) and carotenoids (carotenoids α and β, xanthophylls) through the process of gravity column chromatography.

2. Experimental Section

Ten red peppers were deseeded and chopped. It was then pounded using a mortar and pestle with sand in it. The mixture was then titrated and filtered with 20 ml CH2Cl2. A small piece of cotton was placed inside a 10 ml plastic syringe with the needle tip removed and was replaced by a burette stopper. Slurry of silica in hexane was prepared. Slurry of silica was pipetted quickly using a clean dropper to avoid the silica of drying out. One drop of the chili pepper extract was placed on top of the silica. After allowing it to go down and absorbed another drop of the extract was placed on top of the silica. This process was done several times. No liquid extract was left on top of the silica. The first eluting solvent which was hexane was added to the silica allowing it to go down. The same procedure was done with the second solvent hexane-CH2Cl2, third solvent CH2Cl2 and fourth solvent CH2Cl2-MeOH. The pigments were collected in a clean dry test tube when the color is about to be eluted out of the column. The samples were then covered with aluminum foil and protected from direct sunlight.

3. Results and Discussion

Two eluates were obtained from the extraction of the chili peppers. The first appearance of the yellow solution was in 237 drops and the orange eluate was obtained with 147 drops as shown in Table 1. DCM was used to obtain these solutions. Using the TLC plate the solutions were tested to obtain the numbers of compounds in the solutions. The Yellow solution contained one compound which is considered the pure solution while the Orange one contained three compounds with one compound visible even without the UV.

|Color of Solution Gained |Volume of Eluates in drops |
|Yellow |147 |
|Orange |237 |

Table 1: Column Chromatography Results

In the yellow solution there was only one component and it was visible in UV light, it traveled to 3.8 cm from the starting point. The orange solution had three components, the 1st component traveled 2 cm from the starting point and visible to the eye, the 2nd traveled 2.5 cm from the starting point detected by UV light and the 3rd component traveled to 3.8 cm from the starting point visible in UV light.

| |Distance traveled by the |Distance traveled by the DCM |Retention factor |
| |compound | | |
|Yellow solution | | | |
|Component A |3.8 cm |4.5 cm |0.844 |
|Orange solution | |4.5 cm | |
|Component A |2 cm |4.5 cm |0.444 |
|Component B |2.5 cm |4.5 cm |0.555 |
|Component C |3.8 cm |4.5 cm |0.844 |

Table 2: Thin-Layer Chromatography Results

The table shows the computed Rf with the corresponding values of the Distance traveled by the compound for each component.

Retention Factor is computed by dividing the distance traveled by the compound to distance traveled by the solution. Below is the computation of the Rf values.

Yellow Solution
Component A: Rf= 3.8/4.5= 0.844

Orange solution
Component A: Rf = 2/4.5= 0.444
Component B: Rf = 2.5/4.5= 0.555
Component C: Rf = 3.8/4.5= 0.844 Carotenoids are non-polar hydrocarbons. These are yellow, orange, or red pigments synthesized by many plants, fungi, and bacteria.. In plants, carotenoids are found roots, stems, leaves, flowers, and fruits. Carotenes, xanthophylls, and capsanthin are types of carotenoids. Carotenes consist only of carbon and hydrogen atoms while xanthophylls and capsanthin have one or more oxygen atoms. Carotenes appear yellow-orange; xanthophylls are yellow; and capsanthins are red.

4. Conclusion

The components of capsicum frutescens were separated and identified through analyzing the polarity of each. The colors reflected by the pigments were useful as well in the determination. The yellow component in the TLC is the xanthophylls, with an Rf value of 0.844. Unfortunately, this is the only visible component that was extracted. Possible sources of errors were the manner the column was packed with silica gel or the sequence of the reagents used.

5. References

Gregory, R.P.F. (1989). Biochemistry of photosynthesis (3rd ed.). Chichester : John Wiley & Sons. Ltd.

Heftmann, E. (ed.). (1961). Chromatography : adsorption, partition, ion exchange, electrochromatography, column, slab paper, gas. New York : Reinhold.

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β-Carotene

References: Gregory, R.P.F. (1989). Biochemistry of photosynthesis (3rd ed.). Chichester : John Wiley & Sons. Ltd. Heftmann, E. (ed.). (1961). Chromatography : adsorption, partition, ion exchange, electrochromatography, column, slab paper, gas. New York : Reinhold. ----------------------- β-Carotene