CHROMATOGRAPHY

Lorraine M. Ramos, Sharmaine Michelle M. Reyes, Rhone Arevyn E. Roque, Joseph T. Sabido and Jan Armelynn S. Santos Group 7 2A Medical Technology Organic Chemistry Laboratory

ABSTRACT
Chromatography is an effective technique for separating mixtures. In this experiment, the pigments of red siling labuyo were extracted by using DCM-hexane (1:1). The extract was introduced in the column with silica gel and colored eluates were collected. The eluates were then tested for their purity in the TLC plate inside the developing chamber through the technique of thin layer chromatography. UV lamp was used to visualize the components and retention (Rf) values of the pigments were calculated.

INTRODUCTION
Chromatography is a technique that is widely used for the separation of gases, liquids, or dissolved substances. After being separated, the individual constituents can be identified or purified through standard techniques. Its accuracy and ease of use make chromatography a fundamental procedure in chemistry [1]. Chromatography involves a sample (or sample extract) being dissolved in a mobile phase which may be a gas, a liquid or a supercritical fluid. The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase. Chromatography types such as high performance liquid chromatography, a chromatography type in which the mobile phase is liquid and gas chromatography, a kind of chromatography in which the mobile phase is gas use columns narrow tubes packed with stationary phase, through which the mobile phase is forced. The sample is transported through the column by continuous addition of mobile phase. This process is called elution. The solution of the solvent and the dissolved matter is the eluate. The average rate at which an analyte moves through the column is determined by the time it spends in the mobile phase [2]. Various kinds of chromatography are possible depending on which two phases used. Column and thin layer chromatography involve solidliquid; paper and high performance liquid chromatography involve liquid-liquid and; vapour-phase chromatography involves gasliquid method [3].

In this experiment, solid-liquid phase was used. Therefore, column chromatography and thin layer chromatography were performed. To separate colored components in red siling labuyo with the use of column chromatography; to test their purity with the use of thin layer chromatography and; to measure the Rf value of each colored component are the objectives of this experiment. In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass 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 analysed in column chromatography is applied to the top of the column. The liquid solvent (the eluent) is introduced in the column by gravity or by the application of air pressure. 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 are collected as the solvent drips from the bottom of the column. Silica gel and alumina are the solid adsorbents used in column chromatography. The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column. Polar solvents can more effectively compete with the polar molecules of a mixture for the polar sites on the adsorbent surface and will also better solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar molecules rapidly through the column. If a solvent is too

polar, movement becomes too rapid, and little or no separation of the components of a mixture will result. If a solvent is not polar enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus crucial to the successful application of column chromatography as a separation technique [4]. The solvents used for this experiment are DCMhexane, DCM and DCM-methanol respectively with DCM-hexane as the least polar and DCMmethanol as the most polar. Moreover, silica gel was the adsorbent used.

only the very bottom of the plate is in the liquid. This liquid 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, 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 plate itself contains a fluor which fluoresces everywhere except where an organic compound is on the plate [5].

Figure 1. Column Chromatography On the other hand, thin layer chromatography (TLC) is a simple, fast, and cheap procedure that gives the chemist a quick answer as to how many components are in a mixture. It 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). Figure 2. Thin Layer Chromatography A TLC plate is a sheet of metal which is coated with a thin layer of a solid adsorbent (usually silica or alumina). A small amount of the extract to be analysed 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 The retention factor, or Rf, is defined as the distance travelled by the compound divided by the distance travelled by the solvent.

paper, covered with watch glass and allowed to equilibrate. The larger an Rf of a compound, the larger the distance it travels on the TLC plate. When comparing two different compounds run under identical chromatography conditions, the compound with the larger Rf is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a mixture, you can predict that a compound of low polarity will have a larger Rf value than a polar compound run on the same plate [6]. The TLC plate was then placed in the equilibrated developing chamber. The solvent system was allowed to rise up to 1 cm from the upper end of the plate. The plate was removed from the chamber and dried. Lastly, the components were visualized under the UV lamp and their Rf values were measured.

RESULTS AND DISCUSSION

1. Column Chromatography Four colored eluates were yielded through column chromatography. These are yellow, light yellow, orange and light orange. The first eluate, yellow, has 102 drops that were collected. There were 75 drops of light yellow; 203 drops of orange and; 89 drops of light orange. The third eluate has the most volume while the second eluate has the least volume. Table 1. Table of Chromatography Color of Component 1 Yellow 2 Light yellow 3 Orange 4 Light orange Results in Column Eluate

EXPERIMENTAL
A. Sample used Red siling labuyo For extracting pigments: DCM-hexane (1:1) Solvent system: 2 mL DCM-hexane (1:1) 2 mL DCM 2 mL DCM-methanol (1:1) B. Procedure 1. Column Chromatography Red siling labuyo pigments were extracted by pouring DCM-hexane (1:1) into the siling labuyo in the mortar and pestle and macerating it. The extracted pigments were set aside In a Pasteur pipette, cotton was plugged and silica gel was put uniformly in the column up to its indented part. No air space was allowed to exist. Using another pipette, 0.5 mL of the extract was placed on top of the column. 2 mL of each eluent was introduced in the column. The first eluent was DCM-hexane, second is DCM and last is DCM-methanol. The column was not allowed to run dry. Colored eluates were collected and the colorless eluates were discarded. Drops of each colored eluate were counted. 2. Thin Layer Chromatography The collected eluates were applied on a 5 cm x 8 cm precoated TLC plate by spotting 10 times. Each spot was dried before applying the next. The developing chamber was prepared by placing an amount of DCM-hexane not higher than the level of the origin of the extract (1 cm from the lower end of the plate) in the TLC plate. The chambers inner wall was lined with filter

Volume of (drops) 102 drops 75 drops 203 drops 89 drops

2. Thin Layer Chromatography The yellow eluate travelled a distance of 3.2 cm. The light yellow travelled a distance of 5 cm, the orange with 1.1 cm and the light orange with 3.9 cm. The light yellow and light orange were UV visible. The solvent travelled a distance of 6 cm. Table 2. Table of Chromatography Color of Component Yellow Light yellow Orange Light Orange Results in Thin Layer

Distance travelled by component from origin 3.2 cm 5 cm 1.1 cm 3.9 cm

The light orange component travelled the farthest while the orange component travelled the shortest. In computing for the Rf values of each component, the distance travelled by the component was divided by the distance travelled by the solvent.

Yellow component: 0.53 Light yellow component: 0.83 Orange component:

http://orgchem.colorado.edu/hndbksupport/TLC/ TLC.html 08/13/11 [6] University of Colorado, Boulder, Chemistry and Biochemistry Department. TLC: Retention Factor http://orgchem.colorado.edu/hndbksupport/TLC/ TLCrf.html 08/13/11

Light orange component:

The developed plate wasnt able to show the separation of components well. One possible of error is when spotting the component on the plate, the spot was not completely left dry before placing the succeeding spots. Another is that the spots werent so small enough, causing the colors to disarray. When the developing chamber was not covered completely during the development of the TLC plate can also be a source of error. Therefore, the larger the Rf of a component is, the greater the distance it travelled in the TLC plate. It is also less polar because it reacts less strongly with the polar adsorbent on the TLC plate.

REFERENCES
[1]Chromatography http://www.justchromatography.com/chromatogr aphy 08/13/11 [2]Chromatography http://teaching.shu.ac.uk/hwb/chemistry/tutorial s/chrom/chrom1.htm 08/13/11 [3] Bayquen, A. et al (2007). Laboratory Manual in Organic Chemistry. Quezon City: C&E Publishing, Inc [4] University of Colorado, Boulder, Chemistry and Biochemistry Department. Column Chromatography http://orgchem.colorado.edu/hndbksupport/colch rom/colchrom.html 08/13/11 [5] University of Colorado, Boulder, Chemistry and Biochemistry Department. Thin Layer Chromatography