Practical Protein Chemistry - A. Darbre 1989

Separation of protein and peptide mixtures by high-performance liquid chromatography
Ion Exchange

The Development of ion-exchange Supports suitable for HPLC began with the pioneering work of individual researchers and companies, most notably Pharmacia [17, 18].

Currently, four types of supports are known:

1. Glycophases — Glass particles coated with an epoxy polymer film On the surface, covalently linked to glass silanol groups via glycerylpropyl linkages. They are supplied as diethylaminoethyl, carboxymethyl, and sulfopropyl derivatives, as well as quaternary ammonium derivatives known as CPG glycophases.

2. Pellicular sorbents contain DEAE residues in their side chains. They are supplied by companies as Synchropak columns (Synchrom Incorp.).

3. Ion-exchange sorbents by Toyo Soda (Japan) are Gel Chromatography supports modified for ion-exchange Applications.

4. Supports for FPLC (Pharmacia, Sweden).

These ion exchangers can be used for rapid separations, serving purposes similar to those performed on conventional soft gels. FPLC supports have been successfully employed to fractionate complex mixtures [17].

Anion-exchange columns, particularly Synchropak AX-300, have been thoroughly evaluated. With a protein load of 10 mg on a 4.1 x 250 mm Column at a flow rate of 0.5 mL/min and a sodium acetate concentration gradient (0–0.5 mol/L) in Tris buffer (pH 8) over 90 min, the recovery of various Enzymes ranged from 90% to 100% [29].

Undoubtedly, intensive research into The properties of such sorbents will continue in the coming years, and rapid ion-exchange separations are expected to become standard practice. Currently, the FPLC method is preferred for the high-performance ion-exchange fractionation of peaks obtained via HPLC.