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

Ion-exchange chromatography of peptides
Fractionation of acidic peptides on an Amberlite IR-4B column

Principle of the method. Amberlite IR-4B anion exchange resin, used in the acetate form, binds the strongly acidic peptide fraction at solution pH values between 5 and 6, which can then be eluted in exchange for Cl^- ions.

Scope of application. This resin is primarily used for fractionating small Peptides and isolating acidic Amino Acids and peptides from a mixture.

PROCEDURE

1. Preparation of the resin Column. Approximately 3.0 g of dry resin is required to bind 15 mg of dicarboxylic acid. The resin (200—400 mesh) is suspended in 0.2 N acetic acid and allowed to settle. Unsettled fine resin particles are decanted, and the swollen resin is resuspended, heated to 60° С and stirred for 1 h. After decantation, the resin is washed 4—5 times with distilled Water and heated again in 0.2 N acetic acid to 60° С. The mixture is stirred for 1 h, the supernatant is decanted, and the resin is washed twice with 0.2 N acetic acid. After this, the suspension is washed with distilled water until the pH becomes neutral. The prepared resin suspension is used to pack a 0.5x30 cm column.

2. Chromatography. The material to be fractionated is dissolved such that 15 mg is contained in approximately 30—40 mL of solvent, the pH of the solution is adjusted to 5—6, and the solution is slowly applied to the column. After Sample application, an equal volume of distilled water is passed through the column, followed by elution with 0.1 N НСl. The elution rate should be approximately half the application rate of the fractionated material. The eluate emerging from the column is collected in 1 mL fractions in test tubes. The progress of elution can be monitored using the ninhydrin reaction performed on filter paper aliquots: starting from the 11th tube, 1 drop of each fraction is applied to a sheet of Whatman 3 paper, dried, and developed with a 0.5% solution of ninhydrin in acetone. Elution is continued until no color appears in the collected aliquots.

The obtained fractions are either dried in a vacuum desiccator over КОН or Р₂О₅, or lyophilized.

NOTES

1. By applying gradient elution, further Separation of the obtained fraction of acidic components can be achieved.

2. The resin is regenerated in the same manner as before packing the column.

3. The eluate obtained by washing the column with distilled water after sample application and before elution with 0.1 N НСl contains the neutral and basic Components of the fractionated peptide mixture.