COLUMN AND THIN LAYER CHROMATOGRAPHY

Leanne Camille R. Chin, Arveec C. Chion, John Joebert R. Collado, Claude Ericson C. Coronado, Danielle Wyneth V. Cruz Group 4 2C Medical Technology Organic Chemistry Laboratory

ABSTRACT
[1] Chromatography is a separation method that relies on the differences of partitioning behaviour between a flowing mobile phase and a stationary phase. There are various types of chromatography but in this experiment, column and thin layer chromatography were used. Our subject of extraction are the malunggay leaves, wherein we used hexane:acetone to get the extract of the malunggay leaves. In column chromatography, where three types of eluates were used in order to acquire the multiple pigments in the extract: hexane acetone, acetone and DCM hexane. Silica gel was also used to help us differentiate/separate the colours of the pigments. In thin layer chromatography, eluates and the extract are spotted 10 times on a TLC plate which was placed on a developing chamber containing hexane:acetone. The UV lamp was also used to further visualize the results in the TLC plate. Thus in this experiment, we were able to visualize the pigments of the malunggay leaves.

INTRODUCTION
[3] Chromatography is a very special separation process for many reasons. First of all, it can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography. In fact, chromatography can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed. Second, chromatography can be used to separate delicate products since the conditions under which it is performed are not typically severe. For these reasons, chromatography is quite well suited to a variety of uses in the field of biotechnology, such as separating mixtures of proteins. [2] Chromatography is a technique used to separate a sample into its individual parts. This separation occurs based on the interactions of the sample with the mobile and stationary phases. Because there are many stationary/mobile phase combinations that can be employed when separating a mixture, there are several different types of chromatography that are classified based on the physical states of those phases. In this experiment, we used column and thin layer chromatography. In column chromatography, we have three different eluates (hexane:acetone, acetone and DCM hexane) and a silica gel on the Pasteur pipette to separate the colors of the extract of malunggay leaves. [4] In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column and the mobile phase, a liquid, is added to the top and flows

down through the column (by either gravity or external pressure). Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture. The mixture to be analyzed by column chromatrography is applied to the top of the column. The liquid solvent (the eluent) is passed through the column by gravity or by the application of air pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column. Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved. The individual components, or elutants, are collected as the solvent drips from the bottom of the column. In thin layer chromatography, we spotted the eluates(which are from the column chromatography) and the extract with capillary tubes in a TLC paper and put it in a developing chamber which contains hexane:acetone. [5] TLC is 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 (preferably both run on the same TLC plate). 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, an 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. The objectives of this experiment are: to separate the colored components of malunggay leaves using column chromatography, to determine the purity of the components using thin layer chromatography and to measure the Rf values of the colored components in the thin layer chromatography.

components of the extract. As the colors change, test tubes were changed as well and the number of drops in every color was also noted.

Figure Chromatography

Column

EXPERIMENTAL
A. Samples Used Extract of malunggay leaves B. Procedure 1. Extraction In this procedure, the malunggay leaves are first, cut into pieces; second, 10mL hexane:acetone was poured; lastly, triturated with mortar and pestle in order to get its extract. 2. Column Chromatography The first thing we did was to prepare the Silica Gel in a Pasteur pipette, with a small piece of cotton at the bottom. After plugging the Silica Gel, we let it swell by applying hexane:acetone first and then applying 0.5 mL of the extract on top. We continuously applied the eluates (0.5 mL hexane:acetone, 0.5 mL acetone and 0.5 mL DCM hexane) to the pipette so that the silica gel wouldnt run dry. With this procedure, we were able to separate the different colored

3. Thin Layer Chromatography After collecting the eluates from the column chromatography, thin layer chromatography was performed. In this procedure, we spotted the extract and the eluates on a TLC plate 10 times with capillary tubes. We then placed the TLC plate on a developing chamber which contains hexane:acetone. In this procedure, we can see the pigments of colored components separate. We also observed where the solvent stopped being absorbed. The results were also placed in a UV light for further visualization of the pigments.

Figure 2 Spotting of eluates and extract on TLC plate

Computations of Rf value Rf value = distance of component origin/distance the solvent travelled

from

Light Green Rf = 2.21cm/3.30cm = 0.67 Dark Green Rf = 2cm/3.30cm = 0.61 Lighter than #2 green = 2.1cm/3.30cm = 0.63 Pale green = 2.3cm/3.30cm = 0.7

Figure 3 Chromatography

Thin

Layer

Figure 4 TLC result

RESULTS AND DISCUSSION

From the column chromatography, we were able to get 4 different colored components and are able to measure the volume by the drops. Table 1: Results of column chromatography Color of Component Volume (in drops) Light green 8 Dark green Lighter than #2 green Pale green 7 6 9

REFERENCES
From internet [1] Brian M. Tissue (2000), CHPChromatography Introduction http://www.files.chem.vt.edu/chemed/sep/chromato.html 03/08/13 [2] UC Davis ChemWiki by University of California, Liquid Chromatography http://chemwiki.ucdavis.edu/Analytical_Chemistr y/Instrumental_Analysis/Chromatography/Liquid _Chromatography 03/08/13 [3] Rebecca Carrier and Julie Bordonaro, Intro to Biochemical Engineering Term Project http://www.rpi.edu/dept/chem-eng/BiotechEnviron/CHROMO/chromintro.html 03/08/13 [4] McTony Bio&Chem Inc, Column Chromatography http://www.mctony.com/columnChromatography .html 03/08/13 [5] University of Colorado at Boulder, Department of Chemistry and Biochemistry, http://orgchem.colorado.edu/Technique/Procedur es/TLC/TLC.html 03/08/13

From the thin layer chromatography, we were able to see the pigments of the colored components and the extract and are able to measure the distance from the origin to where the pigments moved. We were also able to measure the distance that the solvent travelled which is 3.30 cm. From these measurements, we were able to get the Rf values of each colored components which are seen in the table below. Table 2: Rf values of each component Color of Distance of Rf value Component component from origin Light green 2.21 cm 0.67 Dark green 2 cm 0.61 Lighter than 2.1 cm 0.63 #2 green Pale green 2.3 cm 0.7