[Recombinant human insulin. VII. Increased efficacy of chromatographic separation based on a principle of...

Retention mechanisms of insulin and deamido[AsnA21] insulin on the bifunctional sorbent Armsphere-C8(PR) in conditions of reversed-phase chromatography (HPLC and ion-pair HPLC) were studied. In accordance with the chemical differences of these proteins, molecular mechanisms of their interaction with silica gel modified with hydrophobic and ion-exchange groups were revealed. The possibility of simultaneous interaction of sorbed proteins with the stationary phase by both mechanisms under conditions of reversed-phase HPLC was demonstrated. The dependences of the separation selectivity and resolution on the mobile phase composition and properties (a salt buffer type, pH, ionic strength) were found. It was demonstrated that the separation selectivity can be regulated by altering the contribution of each of the two separation mechanisms and the bifunctional sorbent used allows higher selectivity in the separation of close protein analogs than monofunctional sorbents.