Separation of peptides and intact proteins by electrostatic repulsion reversed phase liquid chromatography

• An amine group was bonded to the surface of BEH-C18 hybrid particles.
• This material permits the study of electrostatic repulsive interactions chromatography.
• Overloaded band profiles of positively charged peptides and proteins were measured.
• Three columns were made with BEH-C18 bonded with different surface charge densities.
• Three different mobile phase additives were used to purify proteins by gradient elution.

A new brand of BEH-C18 hybrid particles chemically bonded to a leash carrying an amine group permits the implementation of electrostatic repulsive interactions chromatography. Using columns packed with this material, the influence of the concentration of positive charges bonded to the BEH-C18 surface on the overloaded band profiles of a few positively charged peptides and proteins was investigated in the gradient elution mode. Three columns packed with endcapped BEH-C18 particles bonded with three different surface-charge densities (LOW, MEDIUM and HIGH) were used and compared with those provided by a column packed with non-doped, endcapped BEH-C18 particles. The surface concentrations of fixed charges in the LOW, MEDIUM and HIGH columns were estimated at 0.029, 0.050, and 0.064 μmol/m2, for example, about two orders of magnitude smaller than the surface density of bonded C18 chains (2.1 μmol/m2). Three different mobile phase additives (0.1% v/v of trifluoro-acetic, phosphoric, and formic acid) were used to optimize the purification levels of proteins under different loading conditions. The weak ion-pairing ions (formate and phosphate) generate smaller retention but broader, more fronting band profiles than those eluted with a stronger ion-pairing ion (trifluoroactate). This effect is worse in the presence of fixed charges at the surface of the BEH-C18 particles. This was explained by an enhanced anti-Langmuirian adsorption behavior of the charged proteins in the presence of fixed surface charges. As the protein concentration increases in the bulk, so does the internal ionic strength, the electrostatic repulsive interactions weaken, and retention increases. Band fronting is mostly eliminated by replacing weak ion-pairing acids with TFA with which the adsorption isotherm remains weakly langmuirian. Faster but still complete gradient separation of insulin and myoglobin were achieved with the HIGH column than with the reference neutral column, despite a measurable loss in selectivity.