TLC of Phenylthiohydantotn Amino Acids on Silica Gel Layers Impregnated With Metal Salts

Abstract

The determination of primary structure of proteins is still an important and challenging task. The Edman reaction, using phenyl isothiocyanate as the N-terminal labelling agent and making available the N-terminal amino acids as PTH-derivative, lies at the core of all modern sequencing strategies. TLC, however, can be considered to have several advantages over other methods of chromatography because it is sensitive, simpler, and inexpensive, and allows the elution of compounds for subsequent use. The present paper reports the TLC separation and identification of PTH-amino acids in a fifteen component mixture. TLC plates (20×20cm × 0.5mm) were prepared by spreading slurries of silica, gel impregnated with sulphate, acetate, phosphate and chloride of zinc; and sulphates of Mg, Mn, Fe and Co. The chromatograms were developed in pre-equilibrated, rectangular glass chambers using several binary and ternary solvent systems. The spots were located by exposing the chromatograms to iodine vapour. The PTH-aminoacids being weak bases, combine with proton and finally form pairs with available anions (like Cl^-, SO₄.^-, CH₃COO^-) and the solubility and adsorption of the ion pair affects the chromatographic behaviour. The methods are reproducible, simple, require less time and offer resolution of several difficult combinations. The spots are very compact and R_f can be measured very correctly, for identification purpose, in comparison to iodine-azide method where bleached spots with diffused boundaries on a light brown background are located. Attempts were also made to determine the partition coefficients and correlate them with hR_f values.

Key Words:

• PTH-amino acids
• TLC
• Impregnated layers