Column and Thin Layer Chromatography

Categories: Chromatography

Chromatography is a technique used to separate mixtures into components as a result of differential partitioning behavior between a stationary phase and a mobile phase that percolates through the stationary bed. [1, 2]

Column and thin layer chromatography are examples of the solid-liquid method of chromatography, which utilizes an adsorbent solid stationary phase (silica) and a liquid mobile phase. In column chromatography, the solid stationary phase is situated within a tube as the liquid mobile phase is added to the top and allowed to flow down through the tube [3]; in thin layer chromatography, the stationary phase is pre-coated on a plate.

Column chromatography employs both solubility and adsorptivity for the stationary phase.[4] Thin layer chromatography is usually used to determine the purity
of the components of a mixture, and is helpful in determining the identity of an unknown substance based on its Retention factor (Rf) value.[3]

The experiment aimed to: separate the components of malunggay leaves using column chromatography, determine the purity of the components using thin layer chromatography (TLC), and measure the Rf values of the colored components in TLC.

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A. Compounds tested (Samples used)
Sample: Malunggay leaves

Malunggay (Moringa oleifara) is a plant widely known for its nutritional value and herbal uses. It is native to countries with a tropical climate, such as the Philippines, Africa, and India[6], as well as countries in Central and South America. Almost all parts of the malunggay tree may be used for practical reasons, and requires only minimal care for maintenance. For the experiment, malunggay leaves were used as a source for the colored components in both methods of chromatography employed.

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Figure 1. Malunggay leaves

B. Procedure
1. Sample Preparation
Twenty-five pieces of malunggay leaves were titurated using a mortar and pestle, and 5 mL of hexane:acetone (7:3) was added to the sample.
2. Column Chromatography
Filter paper
Filter paper
TLC plate
TLC plate
The set-up for column chromatography was prepared by positioning an iron clamp on the iron stand. The column used was a Pasteur pipette plugged with
cotton which served as a bed support for the stationary phase; silica gel was then uniformly packed into the tube. The column was then clamped onto the set-up. Eluents for the procedure were obtained and placed in test tubes: 7 mL hexane:acetone (7:3), 5 mL hexane:acetone (1:1), 5 mL acetone, and 5 mL acetone:MeOH (1:1). The sample was loaded into the column, followed by the addition of hexane:acetone (7:3) by using a dropper. The colored eluates were collected into test tubes, noting the number of drops per eluate, while colorless eluates were discarded. Without letting the column run dry, hexane:acetone (1:1) was introduced into the column, and in the same manner the eluates were collected. This was the same for the succeeding eluents, and went on until no more colored eluates could be obtained from the column.

Silica gel
Silica gel
Pasteur pipette
Pasteur pipette
Iron clamp
Iron clamp
Iron stand
Iron stand

Figure 2 Column Chromatography
3. Thin Layer Chromatography
The eluates obtained from column chromatography were applied on a TLC plate pre-coated with silica by spotting it seven times per color using a capillary tube. Each spot was dried before applying the next.

A developing chamber was prepared by placing an amount of the solvent system, hexane:acetone (7:3), into a beaker. Filter paper was used to line the walls of the beaker, and was then covered with a watch glass to equilibrate the chamber. Once the filter paper was saturated with the solvent system, the TLC plate was carefully placed in the beaker to develop.

When the solvent system had reached about a centimeter from the upper end of the TLC plate, the plate was removed, and before allowing it to air-dry, the solvent front was marked. Once air-dried, the plate was placed under a UV lamp to visualize the components to determine any additional colors that were invisible without UV light.

Solvent system
Solvent system
Watch Glass
Watch Glass

Figure 3.1 Thin Layer Chromatography

Figure 3.2 Thin Layer Chromatography
In column chromatography, four colored components were obtained: Dark Green, Light Green, Yellow Green, and Yellow. As seen in Table 1, Light Green was the eluate collected the most in terms of number of drops, while yellow was the least collected. Color of Component| Volume of eluate (drops)|

Dark Green| 67|
Light Green| 200|
Yellow Green| 37|
Yellow| 14|
Table 1 Column Chromatography results
In thin layer chromatography, the solvent front was measured to be 8.2 cm. Two more colored components were produced under the UV lamp: pink and bluish-violet. The distances from the origin of every colored component were measured (in centimeters) and the component that travelled the farthest was Yellow, while the component that travelled the shortest was bluish-violet. This can be seen in Table 2. Color of Component| Distance from the
origin (X) in cm| Dark Green| 4.1 cm|

Light Green| 3.6 cm|
Yellow Green| 3.8 cm|
Yellow| 8.1cm|
Pink| 2.6 cm|
Bluish-violet| 1.6 cm|
Table 2 Distance per Component from the origin obtained from thin layer chromatography
The Retention factor (Rf) value was computed for each component by obtaining the quotient of the distance each component travelled from the origin and the distance travelled by the solvent (solvent front).

The Rf value can used to determine the polarity of two different compounds; the one with a larger Rf value is less polar because it has low affinity to the polar adsorbent on the TLC plate. The Rf value can also be used to determine the identity of an unknown compound. If an unknown compound is experimentally proven to have the same Rf value with a known substance, then it is likely that they are the same compound.[5]

The mathematical equation for Rf value is as follows:
Dividing each component’s distance from the origin by the distance travelled by the solvent, the following values were obtained: Color of Component| Rf value|
Dark Green| 0.5|
Light Green| 0.49|
Yellow Green| 0.46|
Yellow| 0.99|
Pink| 0.32|
Bluish-violet| 0.20|
Table 3 Computed Rf values for each component
[1] Brian M. Tissue. Chromatography. Retrieved 8/11/13
[2] Rebecca Carrier and Julie Bordonaro. Intro to Biochemical Engineering 8/11/13 [3] University of Colorado at Boulder, Department of Chemistry and Biochemistry [4] Bayquen, A., Cruz C., de Guia, R., Lampa, F., Peña, G., Sarile, A. and Torres, P. (2009). Laboratory Manual in Organic Chemistry. Manila: C & E Publishing, Inc. [5] University of Colorado at Boulder. Thin Layer Chromatography

[6] Medical Health Guide. What is Malunggay.

Updated: Jul 06, 2022
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Column and Thin Layer Chromatography. (2016, Apr 04). Retrieved from

Column and Thin Layer Chromatography essay
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