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

Ion-exchange chromatography of peptides
Chromatography of basic peptides on an Amberlite IRC-50 column

Principle of the method. Basic Peptides bind to the Amberlite IRC-50 (H⁺) cation exchange resin and can then be eluted by increasing the H⁺ concentration, thereby separating them from neutral and acidic peptides that do not bind to the resin.

Scope of application. Peptide fractionation in biochemical research.

PROCEDURE

1. Resin preparation. Amberlite IRC-50 resin (200–400 mesh) is suspended in 0.2 N acetic acid, allowed to settle, and the supernatant is then decanted. The wet pellet is suspended in 5 volumes of 0.2 N acetic acid and heated to 60° C. After 30 min, the suspension is cooled, the supernatant is decanted, and the heating step in acetic acid is repeated. The resin is then washed with large volumes of distilled Water until a neutral pH is reached.

A 2 x 30 cm Column is packed with the treated resin and washed with distilled water. The gradient elution mixer is filled with 150 ml of distilled water, and the reservoir with 0.2 N acetic acid, and loading of the sample to be fractionated onto the column is initiated.

2. Chromatography. The sample to be fractionated is prepared as described above (see chromatography on Amberlite IR-4B, p. 195), its pH is adjusted to 5–6, and it is slowly loaded onto the column. After loading, the resin is washed with one volume of distilled water, then the gradient elution apparatus is connected, the magnetic stirrer is turned on, and elution is started. The eluate is collected in 1 ml fractions using an automatic fraction collector. The elution process is monitored by the ninhydrin reaction, performed on filter paper using drops of eluate collected from each fraction.

The collected fractions are dried in a vacuum desiccator and analyzed by paper Electrophoresis. Fractions with identical electrophoretic mobility are pooled.

NOTES

1. If the elution of basic peptides occurs too rapidly, i.e., they emerge practically as a single large fraction, the H⁺ concentration gradient should be made shallower. To achieve this, one can either increase the mixer volume or decrease the acetic acid concentration.

2. When washing the column with distilled water after sample loading and before starting acid elution, a highly dilute eluate is obtained, containing the neutral and acidic Components of the peptide mixture.