Unidimensional, sequential separation of PTH-amino acids by high-performance thin-layer chromatography

Abstract

Eighteen of the twenty common protein PTH-amino acids derivatives are separated by continuous multiple development high-performance thin-layer chromatography. The separation is performed on silica gel plates using five development steps with four changes in mobile phase. The derivatives are identified by scanning densitometry, and the total analysis requires less than 1 h. The unseparated derivatives of alanine and tryptophan are baseline resolved in an alternative solvent system. Thus, all twenty of the common PTH-amino acid derivatives may be identified with the proposed method. By using the unidimensional method of development, the high sample capacity of the high-performance thin-layer chromatographic plate is preserved; samples and standards can be run simultaneously to improve the accuracy of identification. The detection limit for the PTH-amino acid derivatives determined by in situ reflectance scanning densitometry at 270 nm was found to be ca. 0.5 ng per spot.