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Chromatography Lab Report Exercise 4

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Exercise 4


Glister Diadem A. Dollera – 201808525

Chem 31.1—Section EF

BS Biology 1


Chromatography is one of the techniques on separating various substance from a mixture of compound. It involves two different immiscible phases in contact with each other—the mobile phase and the stationery phase. The major types of chromatography are the adsorption chromatography and partition chromatography. This experiment was carried out in order for the students to learn the techniques of paper chromatography and Thin Layer chromatography. However, due to lack of materials, only paper chromatography was carried-out. An Extract from Malunggay leaves through acetone was used as the substance to be analyzed using two different kinds of solvent, the 9:1:1 (v/v/v) hexane: ethanol: acetone and 9:1 (v/v) hexane: acetone. Results have shown that the pigments were separated or scattered on the chromatogram. There is more separation of pigments under the solvent of 3-chemical mixture compared to the two chemical mixture. However, its distance was not measured and Rf value was not calculated. It was observed that the pigments become pale-green color and was barely visible or notable from the naked eyes. Most green pigments just lie on the spot and was not moved toward the top. This might have been brought by certain errors on mixing the acetone on the leaf extract. On the other hand, despite of certain lapses, the students were able to learn the techniques of chromatography and apply it on separating substances. Paper chromatography is an example of liquid-liquid partition chromatography. It is used in the rapid analysis of the components of reaction mixtures and as a tentative means of identification.


        One of various techniques on separating compounds in a mixture and identifying unknown substances is called Chromatography. It is widely used in chemistry and biology. All types of chromatography involve two different immiscible phases in contact with each other. The mobile phase is moving and the other phase, stationary phase, is not moving. The basis of chromatography is that molecules are adsorbed onto the stationary phase at different rates, based on solubility. This causes separation of the mixture. (Richards, n.d)

        Furthermore, according to Lab manual, various types of chromatography are possible depending on the physical states of the stationary and mobile phases involved. Adsorption chromatography uses a solid stationary phase and a liquid mobile phase. Separation using adsorption chromatography is governed by surface adsorption phenomena. On the other hand, partition chromatography uses a liquid stationary phase supported on the surface of a solid and a liquid or gas mobile phase which is insoluble in the stationary phase. Partition chromatographic separations may be due to differences in the solubility of the sample in the stationary and mobile phases. In adsorption chromatography, the mixture to be separated is adsorbed on the solid stationary phase over which the liquid mobile phase is allowed to flow. The transfer of the adsorbed compound between the stationary phase and the mobile phase is an equilibrium process.

        This exercise was carried-out in order to enable the students learn the techniques of chromatography and apply chromatographic methods in the separation of the components of a mixture. The activity focuses solely on paper chromatography due to lack of other apparatus. In paper chromatography, a solvent moves from one end of a paper to the other end, as the paper absorbs it. Since it is moving, the solvent is the mobile phase. The paper is the stationary phase. (More specifically, the cellulose molecules in the paper contain many polar –OH (hydroxyl) groups that serve as the stationary phase).


[pic 1]

Scientific Name:  

Moringa oleifera

Common Name:  


Place and Date of Collection:  

Palo, Leyte

Description of Sample:  

Thin, coin-like, green leaves of malunggay plant

Description of Acetone Extract:

Viscous green liquid extract

Table 4.2. Data on the analysis of plant pigments on chromatography

Solvent System

Sketch of chromatogram

[pic 2]

[pic 3]


        According to Richards (n.d), chromatography works by capillary action. In this exercise, a small amount of the leaf pigment extract was first placed near the edge of an absorbent paper. That same edge was wetted with solvent. There were two solvents used. The first solvent was 9:1:1 (v/v/v) hexane: ethanol: acetone. The second solvent was 9:1 (v/v) hexane: acetone. The solvent travels up the paper by capillary action, carrying the spot of leaf extract with it. The distance a chemical travels is determined by its interaction with the solvent (mobile phase) and with the hydroxyl groups attached to the paper. Substances that dissolve more readily in the solvent will move farther than the substances that have a higher attraction for the paper. When the solvent has moved the entire length of the paper, the paper was removed from the solvent and dried. In this case, the leaf pigments is the only chemical involved. However, if there different chemicals involved, they will be located at different positions on the chromatogram.

        The color of the pigment of Malunggay leaves is green. However, the spots on the chromatogram was not that easily distinguished since the pigment appears pale green. This maybe the result of minimal leaf extract mixed with large amount of acetone. According to MSU (n.d), a "pigment" is simply a molecule that absorbs and reflects light. Different pigments appear different colors because they have differing abilities to absorb and reflect various colors of light.  There are different pigments according to color types, and these can be carotenoids, Betacyanins, Anthocyanins, Xanthophylls, Lycopene, Zeathanthin, Chlorophyll, etc. According to Gopi and Elayara (2017), the  pigments present in malunggay leaves are Chlorophyll or green pigment, Carotenoid,  and Xantophyll.



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