Thin Layer Chromatography (TLC): Principle and Procedure


THIN LAYER CHROMATOGRAPHY

PRINCIPLE

       Similar to other chromatographic methods, thin layer chromatography is also based on the principle of separate. The separation depends on the relative affinity of compounds towards stationary and the mobile phase. The compounds under the influence of the mobile phase (driven by capillary action) travel over the surface of the stationary phase. During this movement, the compounds with higher affinity to stationary phase travel slowly while the others travel faster. Thus, separation of components in the mixture is achieved. Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character are identified by means of suitable detection techniques.




PROCEDURE

       The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. These days, however, ready-made plates are preferred.
       With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots.
       Then, samples solutions are applied on the spots marked on the line in equal distances.
       The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom.
        A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect this way).
       Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid.
       The plate is then immersed, such that the sample spots are well above the level of mobile phase (but not immersed in the solvent — as shown in the picture) for development.
       Allow sufficient time for the development of spots.
       Then remove the plates and allow them to dry. The sample spots can now be seen in a suitable UV light chamber, or any other methods as recommended for the said sample



APPLICATIONS

       To check the purity of given samples.
       Identification of compounds like acids, alcohols, proteins, alkaloids, amines, antibiotics, and more.
       To evaluate the reaction process by assessment of intermediates, reaction course, and so forth.
       To purify samples, i.e. for the purification process.
       To keep a check on the performance of other separation processes.

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