EXPERIMENT 5 COLUMN AND THIN LAYER CHROMATOGRAPHY

Myca Pua, Ramon Ramos, Amanda Redilas, Kimleigh Reyes, Nathaniel Sim and Clara Tamondong Group 9 2F Medical Technology Organic Chemistry Laboratory

ABSTRACT
Chromatography is a modern and sophisticated laboratory separation technique, which is used to separate mixtures and to determine their purity. The principle employed in chromatography is the difference of affinity of solute in two phases, the stationary phase and the mobile phase. In this experiment colored pigment extracted from the red Siling Labuyo used with DCM-hexane (1:1) passed through column chromatography to separate its colored pigment. After this process, the eluate collected from the column chromatography passed through thin layer chromatography to determine its purity by computing the Rf (Retention factor) of each colored component seen with and without the UV lamp.

INTRODUCTION
Chromatography is a technique in whichcompounds in a mixture is separated based on differing affinities between a mobile phase and a stationary phase. The mobile phase passes through the stationary phase. Mobile phase is a medium used in chromatography which moves through the stationary phase. In Thin layer chromatography and column chromatography, the mobile phase is an organic liquid. Stationary phase is a material used in chromatography which does not move. The stationary phase is either a pure solid substance such as alumina or silica or a thin coating of liquid on a solid support or a gel. In Thin layer chromatography the stationary phase is a spread in a thin layer on an inert support, usually a plastic sheet or a glass plate. The mobile phase moves upward by capillary action. In this experiment two kinds of chromatography that was used, column chromatography and thin layer chromatography. Column chromatography is primary used to separate relative large samples into pure components. Column chromatography is a preparative technique. In this experiment, Column Chromatography was used to separate the mixture treated with the mobile phase through the column packed silica gel which is the stationary phase. After the mixture is applied to the column packed silica gel, an appropriate solvent is used to help it pass through the column. The different eluent passed through the column with different speed. Silica gel was used for the stationary phase because compounds usually adheres to silica to different extends (Kahn). Thin layer Chromatography is used primarily as an analytical technique. It can be used to identify components of a mixture (by comparing Rf

values), monitor the progress of a reaction (by comparing the intensity of spots) and check the purity of a sample (more than one spot indicates an impure sample). Rarely is TLC used to isolate and purify compounds into discrete samples. In this experiment, thin layer chromatography was used to separate the different components of the eluate obtained from the column chromatography. From this the purity of each component can be computed using the Rf (Retention Factor) value of each component. Rf (Retention Factor) is the ratio between the distance travelled by the solute relative to the distance travelled by the solvent (Ettre), in this case, DCM: Hexane (1:1). Through these processes the following objectives were met, mainly: 1. Separate the colored components of Siling Labuyo leaves using column chromatography. 2. Determine the purity of the components using thin layer chromatography (TLC). 3. Measure the Rf (Retention Factor) values of the colored components in TLC.

EXPERIMENTAL
A. Compounds tested (or Samples used) 5 Siling Labuyo, silica gel, DCM-hexane (1:1), DCM, DCM-methanol B. Procedure 1. Column Chromatography 5 Siling Labuyo was used in the experiment this was prepared by removing the crown, then slicing the Siling Labuyo open to remove the seeds inside. After this the Siling Labuyo was minced to small pieces then grinded using the mortar and pestle to extract the liquid, DCMhexane (1:1) was added to the mixture. After extracting enough amount of mixture from the Siling Labuyo, a portion from the mixture was set aside for Thin layer chromatography.

In one Column, cotton was plug for the purpose of bed support. On top of the cotton, was a uniformly packed silica gel up to the indented part of the column. Silica gel was uniformly packed by tapping the column with another Pasteur pipette and its bulb hitting the column while the silica gel is added. DCM-hexane was then added to the column. Once the set-ups are done 0.5ml of the extracts from Siling Labuyo was added to the column using a Pasteur Pipette. The mixture was then introduced with the first eluent DCMhexane (1:1) making sure that the column does not run dry. The colorless eluate was discarded while the colored eluate was tabulated by number of volume. After using all the DCMmethane (1:1), the second eluent, DCM was added to the solvent system in portions making sure that the column does not run dry, the number of volume is also observed. After the DCM is all used up, the third eluent was added to the solvent system in portions making sure that the column does not run dry, the number of volume is also observed. The colored eluate was placed in separate test tubes for analysis. Number of volume was carefully taking note of. 2. Thin Layer Chromatography A developing chamber was prepared by using one beaker as the jar and an inverted watch glass as the cover. DCM-hexane (1:1) was placed inside the beaker; the inner walls were lined with filter paper to allow the TLC plate to stand. The beaker was covered with watch glass and was allowed to equilibrate. Eluate gathered from the column chromatography is equidistantly spotted 10 times in the 5cm X 8cm recoated TLC plate; each drop spotted slowly allowing the drop to dry before applying the next drop. It was ensured that the spot were small enough so that when the colors separate they wont mix with each other. The TLC plate was then introduced to the developing chamber, allowing the solvent to rise until one cm from the upper end of the TLC plate. Once the plate is removed from the developing chamber the solvent fronts were marked. The components were marked using the help of the UV lamp. Retention factor (Rf) values were computed and the TLC plate was kept for documentation.

RESULTS AND DISCUSSION

Plant used: Siling Labuyo Solvent system used: DCM- hexane (1:1) Column Chromatography Three colored eluate were obtained from the extraction of Siling Labuyo, namely yellow, light yellow and orange. A fourth color emerged which is dark orange but the amount of elate gathered was too small and wasnt enough for spotting.
Color of Component 1 2 3 4 Yellow Light Yellow Orange Dark Orange Volume of eluate (drops) 78 drops 52 drops 34 drops 3 drops

Table 1 Column Chromatography (Table Results) Thin Layer Chromatography

Figure 1 Red Siling Labuyo TLC plate Figure 1 shows the spotting spot 1 is the Yellow spot, spot number 2 is the light yellow spot and spot 3 is the orange spot. The yellow component traveled 5.9 cm from the starting point. The light yellow component travelled 4.8 cm, while the orange component travelled 4.8 cm. The final plate was placed under UV lamp to look for spots not visible to the naked eye.

Calculation of Rf (Retention Factor) The Rf in TLC is the ratio of the distance the spot traveled to the distance the solvent traveled on a TLC plate, the higher polarity the compound, the larger affinity of the compound to the stationary phase and the smaller the Rf, the lower the polarity the compound, the higher the affinity to the solvent and the larger the Rf. If a solvent is changed from a low polarity solvent to a higher polarity the eluting power will increase and all the Rf values will increase. To obtain an Rf measure the distance from the solvent start line to the middle of the spot. The distance between the smallest two lines on the ruler should be approximated. Computations:

From the internet [1]http://www.sigmaaldrich.com/etc/medialib/do cs/SigmaAldrich/Product_Information_Sheet/ a1772pis.Par.0001.File.tmp/a1772pis.pdf (August 12, 2012) [2]Fisher Scientific Catalogue Product Specifications http\\www.fisher.sci.com (August 12, 2012) [3]Fisher Scientific MSDS sheet Ferrocene http://fscimage.fishersci.com/msds/03388.ht m and Acetylferrocene (August 12, 2012) [4]https://fscimage.fishersci.com/msds/69220.ht m (August 12, 2012) [5]Fisher Scientific MSDS for Dichloromethane methylene chloride MSDS (August 12, 2012) [6] http://www.rpi.edu/dept/chem-eng/BiotechEnviron/CHROMO/chromintro.html (August 12, 2012)

yellow

light yellow

orange

1 2 3

Color of Component Yellow Light yellow Orange

Distance 5.9 cm 4.8 cm 4.8 cm

Rf 0.95 0.77 0.77

Table 2 Thin Layer Chromatography and Rf value

REFERENCES
From books [1] Experimental Organic chemistry, 2nd Ed, J. C Gilbert & S. F. Martin, Sanders New York 1994, P157 [2] Quantitative Chemical Analysis, D. C. Harris W.H Freeman 1982 p584 [3] Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43(14), 29232925. doi:10.1021/jo00408a041