COLUMN AND THIN LAYER CHROMATOGRAPHY Dan Neil Fabian, Jomarie Galao, Harold Vincent Go, Patricia Margarita

Guadines and Christine Evan Ho Group 4 2F Medical Technology Organic Chemistry Laboratory ABSTRACT
Chromatography is a method of separating mixtures that are used for solid-liquid, liquid-liquid, and gas-liquid mixtures. In this experiment, pigments of the malunggay leaves were extracted with the use of Hexane-Acetone, extract was introduced into the column and eluate was collected, this process is the Column Chromatography (CC) method. The purity of the components was determined with the use of Thin-Layer Chromatography (TLC). UV lamp was then used to visualize the developed TLC plate and the Retention or Retardation Factor was measured.

INTRODUCTION
Chromatography can be defined as a modern laboratory technique that separates a mixture into its various components by using the differential solubility or adsorptivity of the components to be separated for a stationary medium and for a mobile medium through which they pass. The principle underlying chromatography is that the different substances have also different partition coefficients between the stationary and mobile phases. Compounds that interact weakly with the stationary phase will move rapidly through the chromatographic system. While, compounds that interact strongly with the stationary phase will move slowly through the chromatographic system. Each component will move through the system at a different rate, resulting in complete separations. Different types of chromatography are possible depending on the two phases used. Gas Chromatography or Vapor Phase Chromatography is the term used when gas is employed as the mobile phase. Separations using gas chromatography involve vapor phase against adsorption and/or equilibria. Liquid Chromatography leads to any chromatographic process that uses a mobile liquid phase. Chromatography separates substances into its components, which is useful as substances are usually unique in their compositions. It can identify a substance and show how it differs from others that may look the same on the surface. Every type of chromatography is useful for analytical purposes. Under specific conditions, all types of chromatography can

be used for preparative scale separations. And in each type, there are elements to be considered; the size of the sample, relative separation of components, and the speed. It would be best if all the elements could be maximized for the quick completion of separation in samples of any size. Generally two of these elements can be maximized at the expense of the third element. For daily analytical work, resolution and speed are maximized at the expense of the load. In preparative scale separations, load and speed can be maximized, but then separation techniques are often incomplete. Complete separations in large samples can be obtained but the overall process is more likely to be slow with the possible involvement of large quantities of solvent that must be distilled for reuse, or discarded. In the experiment, Column Chromatography were used. and Thin

Column chromatography is a technique in which a column of stationary phase is used based on the stationary phase nature; this column chromatography is further divided into two distinct types. If a column of solid stationary phase is used it is called as column adsorption chromatography and if a column of liquid stationary phase is used then it is called as column partition chromatography. Column adsorption chromatography is widely used. Column chromatography is very advantageous over most other chromatographic techniques

because it can be used in both analytical and preparative applications. Not only does column chromatography determine the number of components in a mixture, but it can also separate and purify substantial quantities of those components for subsequent analysis. This is in contrast to paper chromatography which is just an analytical method. The disadvantage of a column chromatography is that it is very timeconsuming, especially for large amount of samples. If unnecessary to prepare a separate large quantity of sample, paper chromatography is easier to perform. Thin Layer Chromatography uses the same principles as Column Chromatography. The adsorbent is coated on one side of a strip or plate of glass, plastic or aluminum. Then, the solvent travels up by plate through capillary action. Thin-Layer Chromatography is very advantageous because it is simple, fast, and efficient. It also requires small amounts of sample. TLC is very useful for determining the best solvents for a column chromatographic separation. It can be used for an initial check on the identity of an unknown sample. Preparative plates can be carried out with special thick-layered TLC plates. Hexane-Acetone is the solvent system used to elute through a chromatography column. The mobile phase (solvent system) consists of 7:3 (ratio of volume) mixture of hexane (C6H14), and acetone (C3H6O). The solid phase is eluted with this solvent system, the compound to be purified is then loaded onto the solvated solid phase, and the column is eluted with the same solvent system until desired compound has come off the column. The Retention or Retardation Factor (R value) is the ratio of the distance that the spot has travelled relative to the distance moved by the solvent which in this case is the Hexane-Acetone.

The objectives of the said experiment are the following; separate color components in malunggay leaves using column chromatography, determine the purity of components using thin-layer chromatography, and measure the Retention factor values of the colored components in thin-layer chromatography.

EXPERIMENTAL
A. Compounds tested Plant used: Malunggay leaves Solvent-system used: Hexane-Acetone (7:3) B. Procedure Pigments of Malunggay leaves were extracted using Hexane-Acetone (1:1) and eventually pounding it using mortar and pestle. The extracted pigments were set aside for a while. 0.5 mL of the extract was placed on top of the column using a Pasteur pipette. The pigments wre eluted using 10 mL of Hexane-Acetone (7:3) and the solvent system was introduced in portions. The column was not allowed to run dry and the colorless eluate was discarded. The test tubes were changed each time the color of the eluate varies. The number of drops for each color was noted. After collecting the eluates from the column, Thin-Layer Chromatography was performed. The eluates were applied on a 5x8 cm pre-coated TLC plate by spotting it 10 times as small as possible. The spots were allowed to dry before applying the next. The developing chamber was prepared by putting an approximate amount of HexaneAcetone (solvent system) on a beaker. The inner chamber was covered with a watch glass, and was allowed to equilibrate. Thin-Layer Chromatography was introduced in the developing chamber allowing the Hexane-Acetone to rise up until it reaches the mark of 1 cm before the upper end of the plate. The solvent front was spotted and was air-dried.

The components were viewed under the UV lamp to be visualized, then R values were measured.

the solvent front; the first component travelled 4.50 cm, the second component travelled 5.90 cm, the third component travelled 4.00 cm. Computation of R values: R = distance travelled by solute/ distance travelled by solvent R first component = 4.5/5.9 = 0.76 The computed R value of the component was 0.76. R second component = 5.9/5.9 = 1.0 The computed R value of the component was 0.76. R third component = 4.0/5.9 = 0.68 The computed R value of the component was 0.76. first

RESULTS AND DISCUSSION

In Column Chromatography, four eluates were produced in the presence of yellow, dark green, yellow green and pale yellow. Table 1. Color of components obtained and their respective volumes
1 2 3 4 Color of the Component Yellow Dark green Yellow green Pale yellow Volume of eluate (drops) 30 drops 40 drops 42 drops 44 drops

first

Table 1 shows how many eluates were produced and their respective volumes. The first eluate, yellow, yielded 30 drops. The second eluate, dark green, yielded 40 drops. The third eluate, yellow green, yielded 42 drops. And the last eluate, pale yellow, yielded 44 drops. In Thin-Layer Chromatography, the R values of the crude extract and the eluates from column chromatography was computed based on the distance travelled by the spot divided by the spot travelled by the solvent front. Since R is a ratio, it has no unit of measurement. The Thin-Layer Chromatography produced three different colors from the crude extract and the eluates from Column Chromatography. These were green, yellow, and yellow green colors. Table 2. Components obtained and their distances from the origin
Color of the Component 1 2 3 Green Yellow Yellow green Distance of component from origin (cm) 4.5 cm 5.9 cm 4.0 cm R value 0.76 1.00 0.68

first

The computed values signify the ratio of the distance travelled by the center of a spot to the distance travelled by the solvent front. In generally, it is the fraction of an analyte in the mobile phase of a chromatographic system.

REFERENCES
From books University of Santo Tomas Faculty of Pharmacy, Organic Chemistry Group. (2009). Laboratory Manual in Organic Chemistry. Quezon City: C&E Publishing, Inc. From the internet Sandhyasravya, M. Column Chromatography http://www.pharmainfo.net/msandhyasrav ya/blog/column-chromatography Column Chromatography http://www.chem.ubc.ca/courseware/121/ tutorials/exp3A/columnchrom/ http://ww w.chemguide.co.uk/analysis/chromatograp hy/column.html#top 8/15/12 Thin Layer Chromatography http://chemistry.csudh.edu/faculty/noel/C HE317L/Thin%20Layer%20Chromatorgaph y%20Experiment.html 8/15/12

The distance travelled by the solvent was 5.90 cm from the origin of the TLC plate to