Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Thin-Layer Chromatography of Amino Acids
Separation Methods

Thin-Layer Chromatography (TLC) is the simplest and most accessible method for amino acid Separation, though its quantitative capabilities are quite limited. Individual spots can be identified only by carefully comparing chromatograms with those of standard Amino Acids separated simultaneously under identical conditions. Procedures for preparing silica gel and Cellulose thin-layer plates, as well as conditions for two-dimensional amino acid separation, have been described [44]. Several manufacturers produce TLC plates that provide reproducible results.

1. The separation of 24 amino acids, including leucine and isoleucine, on 200x200 mm plates coated with a 100 µm layer of MN-300 cellulose (Macherey Nagel & Co.) has been described [190]. The total analysis time is 6 h.

Solvent for the first dimension: propan-2-ol—formic acid—Water (40:2:10); for the second dimension: tert-butanol—methyl ethyl ketone—0.88 NH₃—water (50:30:10:10).

2. An improved solvent system has been proposed that allows the separation of 40 Amino Acids and related compounds on binder-free MN-300 cellulose plates [147]. Solvent for the first dimension: propan-2-ol—2-butanone—1 M HCl (60:15:25); for the second dimension: 2-methylpropanol—2-butanone—propanone—methanol—water—0.88 NH₃ (40:20:20:1:14:5). Plates are washed prior to use to remove impurities. The chamber must be saturated with each component of the solvent mixture. After development in the first direction, the plate is dried with cold air for 15 min, followed by 15 min at 60 °C to remove traces of HCl before second-dimension chromatography. The solvent run time in each direction is 2.5 h. Fig. 8.3 schematically illustrates the separation of a 24-amino-acid mixture.

The same solvent system was used to separate 76 N-containing metabolites found in biological fluids; identification was performed using selective chromogenic Reagents [149] (Section 8.14).

3. Avicel thin-layer plates (Analtech) were evaluated for one- and two-dimensional amino acid separation across 6 solvent systems. R_f values for 35 Amino acids are provided. In preparative experiments, sample loads up to 0.05 M were employed [87].

4. The separation of 19 amino acids was performed on reversed-phase thin-layer plates, including those with C₁₈ chemically bonded to silica gel [338].

5. The separation of 24 amino acids on tin tungstate is of some interest, although this method offers no distinct advantages over other techniques [272].