Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Thin-Layer Chromatography (D. A. Medgyesi)
Thin-Layer Chromatography of Amino Acids and Their Derivatives
Peptides

Several authors have successfully used Thin-Layer Chromatography to analyze enzymatic protein hydrolysates [3, 7, 16, 17]. Peptide maps can be obtained by two-dimensional chromatography in the following systems: chloroform—methanol—25% NH₄OH (2:2:1) and pyridine—acetic acid—butanol—Water (40 : 14 : 68 : 25) (double chromatography).

Kieselgel S silica gel (Macherey-Nagel, FRG) is recommended as the support. Prior to the experiment, the silica gel layer is heated at 110°C for 30 min, and this heating is repeated after the first Separation. Most commonly, thin-layer chromatography is combined with Electrophoresis.

Typically, a peptide map can be obtained in 5–10 h. Richard [14], analyzing the tryptic hydrolysate of Myosin by thin-layer chromatography on silica gel (Kieselgel G), used prolonged horizontal chromatography in a chloroform—methanol—34% NH₄OH (2 : 2 : 1) mixture in The First stage. In the second separation stage, electrophoresis was carried out in a pyridine—acetic acid—water (1 : 10 : 489) mixture for 1 h at a voltage of 980 V. Balyo et al. [3] analyzed the tryptic hydrolysate of reduced and alkylated human IgG using thin-layer chromatography on Kieselgel G silica gel (Merck, FRG) containing 0.5% amylopectin. After preparation of the chromatographic plate, the support layer was air-dried. Prior to the experiment, the plates were stored in a desiccator and then used without preheating. In the first separation stage, continuous flow chromatography was applied in a propanol—ethanol—concentrated ammonia—water (20 : 60 : 2 : 20) mixture for 7–8 h. Thereafter, the plate was dried at 60°C and subjected to electrophoresis in the same buffer solution used by Richard [14] for 50 min at a voltage of 1000 V.