OrgChem Formal Report

EXPERIMENT 5 COLUMN AND THIN LAYER CHROMATOGRAPHY

Venneth P. Pascual, Maria Philina Rita C. Pasicolan, Paolo Miguel F. Pineda Christian E. Pontanares and Christelle M. Primaleon Group 8 2F Medical Technology Organic Chemistry Laboratory ABSTRACT Chromatography is a powerful and sophisticated technique in separating solid-liquid, liquid-liquid and gas-liquid mixtures as well as in determining their purity. In this experiment, colored pigments of malunggay leaves were extracted with the use of hexane-acetone and eluates were collected through the Column Chromatography. Thin layer chromatography was then used to determine the purity of the components. The developed Thin layer chromatography plate was seen through the UV lamp. The Retention factor of each colored component was computed.

INTRODUCTION Chromatography is defined as a modern separation technique which relies on the differential solubilities or adsorptivities of the components to be separated with respect to two phases, one being stationary and the other, mobile. The stationary phase is the part of the chromatographic system through which the mobile phase flows where distribution of the solutes between the phases occurs. It is the substance fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography. It often contains a substance which fluoresces in UV light. The more stationary phase is in a column, the more the solutes will be retained, the further they will be apart and the greater the separation. The mobile phase is the part of the chromatographic system which carries the solutes through the stationary phase. The mobile phases are either liquids or gases. The mobile phase consists of the sample being separated or analyzed and the solvent that moves the sample through the column. It is used to adjust the chromatographic separation and retention in liquid chromatography. Various types of chromatography are possible, depending on which two phases are used. These are solid-liquid (column, thin layer), liquid-liquid (paper, high performance liquid), and gas-liquid (vapour-phase) chromatographic methods. In this experiment, column and thin layer chromatography were used. Column chromatography works on a much larger scale by packing the same materials into a vertical glass column. This type can be used not only to determine the number of components of a mixture but also to separate and purify substantial quantities of those components for

subsequent analysis. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose (blotter paper). 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. The liquid or the mobile phase slowly rises up the TLC plate by capillary action. 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. Once visible, the Rf value, or retention factor, of each spot can be determined to compute the purity of each component by dividing the distance the substance travelled by the distance the solvent front travelled using the initial spotting site as reference. These values depend on the solvent used and the type of TLC plate and are not physical constants. The objectives if the experiment were (1) to separate the colored components of malunggay leaves using column chromatography, (2) to determine the purity of the components using Thin layer hromatography and, (3) to measure the Rf values of the colored components of Thin layer chromatography.

EXPERIMENTAL A. Compounds tested (or Samples used) Malunggay leaves, Hexane-Acetone (7:3), silica gel, DCM, DCM-methanol

B. Procedure 1. Column Chromatography

5

Pigments of malunggay leaves were extracted using mortar and pestle and by adding Hexaneacetone (7:3). A portion of the extracts was set aside for Thin layer chromatography (TLC). Cotton was plugged in the column which served as bed support. The column was uniformly packed with silica gel up to the intended part of the Pasteur pipette. 0.5 mL of the extract was placed on top of the column using a Pasteur pipette. The pigments were 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. The colorless eluate was discarded while the colored eluates were colletcted in different test tubes and the number of drops per eluate was noted.

Yellow

65

Table 1. Color of components produced and their respective volumes Table 1 shows how many eluates were produced and their corresponding volumes. The first eluate, yellow green, yielded 64 drops. The second eluate, light yellow, yielded 67 drops. The third eluate, deep yellow, yielded 61 drops. The fourth eluate, light green, yielded 63 drops and the fifth eluate, yellow, yielded 65 drops. The TLC plate produced five different colors from the crude extract and the five eluates from column chromatography. These were yellow green, light yellow, deep yellow, light green and yellow colors.

2. Thin Layer Chromatogrpahy Eluates were slowly spotted 10 times on a 5 cm x 8 cm precoated TLC plate. Each spot was allowed to dry before the next was applied. The spots were applied as small as possible to prevent the mixing of colors. Developing chamber was prepared by putting an approximate amount of hexane-acetone (solvent system) on a beaker. The inner chamber was lined with filter paper for the TLC plate to be able to stand, was covered with an inverted watch glass, and was allowed to equilibrate. TLC plate was introduced in the developing chamber allowing the hexane-acetone to rise up until it reaches the mark of 1cm before the upper end of the plate. The plate was removed from the chamber and the solvent front was marked and was air-dried. The components were viewed under the UV lamp to be visualized. Then, Rf values were measured. The TLC plate was documented.

RESULTS AND DISCUSSIONS Plant used: Malunggay leaves Solvent-system used: Hexane-Acetone (7:3) Column Chromatography In the column chromatography, five eluates were produced mainly yellow green, light yellow, deep yellow, light green and yellow.

1 2 3 4

Color of Component Yellow green Light yellow Deep yellow Light green

Volume of Eluate (drops) 64 67 61 63

Figure 1. TLC plate after introducing to the developing chamber In thin layer chromatography, the Rf values of the crude extract and the five eluates from column chromatography was computed based on the distance traveled by the spot divided by thedistance traveled by the solvent front. Since Rf isa ratio, it has no unit of measurement. The solvent traveled 6.40 cm from the origin of the TLC plate to the solvent front; The first, fifth and eighth component travelled 3.90 cm; the second travelled 2.20 cm; the third travelled 3.58 cm; the fourth travelled 1.79 cm; the sixth travelled 0.08 m, the seventh travelled 0.70 m; the ninth travelled 4.35 cm; the tenth travelled 1.47 cm and the tenth travelled 4.10 cm.

Computation of Rf Values:

Rf =

3.90cm 6.4cm Rf = 0.01

first, fifth and eighth

Rf = 2.20 second component 6.4 cm Rf = 0.34 Rf = 3.58 6.4 Rf = 0.56

third component

Rf = 1.79 fourth component 6.4 Rf = 1.79 Rf = 3.90 sixth component 6.4 Rf = 0.53 Rf = 0.70 seventh component 6.4 Rf = 0.11 Rf = 4.35 ninth component 6.4 Rf = 0.68 Rf = 1.47 tenth component 6.4 Rf = 0.23 Rf = 4.10 eleventh component 6.4

1 2 3 4 5 6 7 8 9 10 11

Component First Second Third Fourth Fifth Sixth Seventh Eighth Ninth Tenth Eleventh

Distance 3.90 2.20 3.58 1.79 3.90 0.08 0.70 3.90 4.35 1.47 4.10

Rf 0.01 0.34 0.56 0.28 0.01 0.53 0.11 0.01 0.68 0.23 0.64

Table 2. The components produced and their distance from the origin.

REFERENCES From books [1] University of Santo Tomas Department of Chemistry. (2006). Organic Chemistry Laboratory Manual. Manila. Pg 57 - 59 From the Internet [1] Retrieved August 13, 2012 http://www.chromatographyonline.org/topics/stationary/phase.html [2] Retrieved August 13, 2012 http://www.chromatographyonline.org/topics/mobile/phase.html [3] Retrieved August 13, 2012 http://www.chemguide.co.uk/analysis/chrom atography/column.html [4] Retrieved August 13, 2012 http://orgchem.colorado.edu/Technique/Proc edures/TLC/TLC.html [5] Retrieve: August 13, 2012 http://www.chemguide.co.uk/analysis/chrom atography/thinlayer.html