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

Thin-Layer Chromatography on silica gel or Cellulose is used to fractionate Amino Acids. While silica Gel chromatography is more sensitive for Certain amino acids than cellulose chromatography, thin-layer cellulose offers superior Separation. Consequently, the method of von Arx and Neher [2] for amino acid separation by thin-layer chromatography on cellulose is presented below.

Preparation of the adsorbent layer. 8.0 g of cellulose powder MN 300 (Macherey-Nagel, FRG) is suspended in a homogenizer in a mixture of 48 ml of distilled Water and 2 ml of ethanol. The resulting suspension is sufficient for coating 5 plates with a layer thickness of 0.25 mm. The cellulose-coated plates are left overnight to dry at room Temperature.

For two-dimensional separation, 4 solvent mixtures are used in three different combinations: 1) n-butanol—acetone—diethylamine—water (10 : 10 : 2 : 5), 2) isopropanol—formic acid—water (40 : 2 : 10), 3) sec-butanol—2-butanone (methyl ethyl ketone)—dicyclohexylamine—water (10 : 10 : 2 : 5), 4) phenol—water (75 : 25) in an atmosphere of 3% NH₄OH.

Solvent (1) is always used for separation in the first direction, and any of the other three Solvents In the second direction. Fractionation is carried out in a standard chromatographic chamber without lining its walls with solvent-soaked filter paper. The solvent is poured into the chamber immediately before the run. The solvent level should be 15–20 mm below the starting point. Chromatography is continued until the solvent front reaches the upper edge of the plate. The plate is then dried for 5–10 min at 90°C or for 12 h at room temperature. In the Second Stage of separation, a standard mixture of amino acids is applied next to the starting point, and the plate is placed in a chamber with the second solvent mixture. After chromatography is complete, the plate is dried at 90°C for 20 min.

It is recommended to develop the resulting chromatograms with a ninhydrin-collidine reagent (1.0 g of ninhydrin dissolved in a mixture of 700 ml of ethanol, 29 ml of 2,4,6-collidine, and 210 ml of acetic acid). Staining with this reagent allows the detection of the following minimum amounts of amino acids: 0.05 μg for Ala, Asp, and Val; 0.1–0.2 μg for Ile, Ser, and Arg; 0.3–0.5 μg for His, Lys, Met, and Tyr.

In The First stage of separation on a thin layer of cellulose MN 300, Nybom [13] performed Electrophoresis for 15 min in 0.7% formic acid at a voltage of 400 V, followed by chromatography in the perpendicular direction in the second stage using a butanol—acetic acid—water mixture (4:1:2).