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
DNP-amino acids

Silica gel has proven to be an excellent support for Thin-Layer Chromatography of DNP-Amino Acids.

Approximately 25—30 g of silica gel is suspended in 60—65 mL of distilled Water and ground in a porcelain mortar until a homogeneous mass is obtained. This amount of silica gel is sufficient for 5 plates measuring 20 x 20 cm with a layer thickness of 0.25 mm. If a support containing calcium sulfate (Kieselgel G) is used, grinding and coating take no more than 1—1.5 min. Silica gel without calcium sulfate (Kieselgel H) yields a stable layer up to 0.25 mm thick. For chromatography of Amino Acids and their derivatives, plates dried in air without heating are used.

Several different Procedures have been proposed for the fractionation of DNP-amino acids. Water- and acid-soluble DNP-Amino acids can be separated by one-dimensional chromatography using a mixture of n-propyl alcohol and a 37% NH₄OH solution (7 : 3, by volume) as the solvent. The sample is applied to the plate in a solution of acetic acid or 0.5 N HCl; however, the acid must be removed before chromatography begins. To achieve this, the chromatographic plate is heated to 60°C prior to application, and the mixture to be separated is applied to the hot support.

Ether-soluble DNP-amino acids can also be separated by two-dimensional chromatography on silica gel using the following solvent systems: 1) toluene — pyridine — Ethylene chlorohydrin — 0.8 N NH₄OH (100 : 30 : 60 : 60), where the upper phase is used as the eluent during chromatography and the lower phase is used for pre-Treatment of the support layer; 2) chloroform — benzyl alcohol — acetic acid (70 : 30 : 3); 3) benzene — pyridine — acetic acid (80 : 20 : 2); 4) chloroform — methanol — acetic acid (95 : 5 : 1).

In The First stage of Separation, system (1) is used. The side walls of the chromatographic chamber are lined with filter paper moistened with the lower phase of this solvent. Two Glass plates are coated with silica gel and placed in the chamber so that the gel layer faces the filter paper but does not contact it.

The plates are left in the chamber overnight, then removed, and the adsorbent layer is immediately covered from above with another glass plate in such a manner that a 15 mm wide strip remains exposed at one end of the first plate. The material to be fractionated is applied to this strip as quickly as possible, and the plate is immediately immersed in the solvent [upper phase of system (1)]. After the solvent front has traveled 15 cm, the plate is removed from the chamber, air-dried for 3 min, and immersed again in the same solvent. After the second chromatography run in the same direction, the plate is dried in a stream of air for 10 min, placed in an oven at 60°C for 10 min, and then cooled in an air stream for 10—15 min. Immediately after this, separation is carried out in the perpendicular direction. When solvent systems (3) and (4) are used as eluents, the prolonged horizontal chromatography takes 2—3 h (see Fig. 46).