Experiment.13 Amino acid analysis by adsorption thin layer chromatography (adsorption TLC)

1.Aim and request Master the basic principle and manipulative technique of adsorption TLC.

2.Principle: Thin layer chromatography is based on the separation of a mixture of compounds as it migrates with the help of a suitable solvent (mobile phase) through a thin layer of adsorbent material which has been applied to an appropriate support (stationary phase).

In adsorption TLC the sample is continually fractionated as it migrates through the adsorbent layer. Competition for active adsorbent sites between materials to be separated and the developing solvent produces continuous fractionation. A portion of the material to be separated will be found in the mobile phase and a portion will be adsorbed to the solid adsorbent particles.

As the process continues the various components move different distances, depending on their relative affinities for the adsorbent as compared with the migrating solvent. In general, the more polar compounds are held back by the adsorbent while less polar material advance further. Polar solvent effects the most movement of sample material over the adsorbent.

3.Procedure: ⑴ With three capillary pipettes, spot a certain volume of Leu solution 、 Lys solution and a mixture solution of Leu 、 Gly and Lys onto the TLC plate, respectively, as shown.. Make sure the spots remain smaller than 2 mm in diameter. Dry the spots With electric blower （ cold wind ）. ⑵ Repeat the manipulation. ⑶ Place the plate in the chamber to develop. Make sure that the loading area is above the solvent

⑷ Immediately close the cover and let run for several minutes, until the solvent front has reached the position about 1cm below the top of the plate. ⑸ Remove the plate from the chamber. Mark the solvent front, then dry the plate with electric blower （ hot wind ）. ⑹ Amino acids separated can be visualized by their color.

4.Results: Solvent front Sample origin lys Leu mixture Leu Gly Lys

Calculate the Rf value Rf = Distance of the amino acid from the origin Distance of the solvent front from the origin Rf=Rf= D1D1 D2D2

Discussion