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

Ion-Exchange Chromatography in a Fixed Ion-Exchanger Bed
Application of “Fixion 50x8” Plates

When using Fixion 50 x 8 plates, the following should be kept in mind:

A. In ongoing studies, such as the Separation of Amino Acids, it is necessary to provide conditions (buffer solution, molarity, pH, Temperature) similar to those used in Column Chromatography, since the functional component of the layer is the same material used for column chromatography, i.e., a strong cation exchanger.

B. Well-established Methods of Thin-Layer Chromatography developed previously can be applied for chromatography, drying, Sample application, visualization, evaluation of separation, documentation, etc. For example, to separate basic amino acids in an amino acid analyzer, a buffer with a Na⁺ concentration of 0.35 M and pH 5.23 is used; this same buffer is suitable for separating basic amino acids on a Fixion 50 x 8 plate. In addition to basic amino acids, aromatic amino acids like Tyr and Phe are also well separated and identified.

When separating basic and aromatic amino acids, the operations are carried out in the following sequence:

1. Mark the start line on the plate using a soft pencil (approximately 1.5 cm from the bottom edge).

2. Apply the test sample using a capillary. If a relatively large volume of a dilute solution needs to be applied, use a hairdryer for drying.

3. Place the plate into a sealed chromatographic chamber with a 1 cm layer of buffer solution at the bottom.

4. Perform chromatography at room temperature until the solvent front rises to a height of 15 cm.

5. Remove the plate from the chamber and dry it with a hairdryer.

6. Spray the plate with a solution of ninhydrin in acetone and dry it with a hairdryer. Heating the plate for a few minutes facilitates the appearance of spots.

In some cases (for example, when separating a mixture of 16 amino acids), the Fixion plate must be equilibrated with a buffer solution before use. During equilibration, just as in column chromatography, the appropriate ionic environment is established and any potential impurities present in the layer are removed. Buffer solutions should be prepared using deionized Water.

Pour 100 ml of buffer solution into a standard-sized chromatographic chamber; the liquid layer at the bottom should be 1–1.5 cm high. This amount of buffer solution is sufficient to perform chromatography on 3 plates. You can place 2–3 plates simultaneously into a single chamber.