pH-dependent elution profiles of selected proteins in HPLC having a stationary phase modified with pH-sensitive...

An approach to controlling protein interactions with silica (glass) beads grafted with pH-sensitive copolymers of a sulfonamide and N,N-dimethylacrylamide is proposed. The bead surface was modified with poly(N.N-dimethyl acrylamide-co-methacryloyl sulfadimethoxine), which exhibits hydrophobic/hydrophilic switching around pH 7.0. Phase transition in the aqueous solution, the wettability change of the modified surface and surface tension of the polymer-grafted glass surface was characterized by changes in turbidity and the dynamic contact angle as a function of pH. The discontinuous phase transition in the copolymer solution shifted to a higher pH region as the composition of sulfadimethoxine monomer (SDM) was increased. However, the transition pH in continuous wettability changes on the modified surface was not affected by polymer composition due to differences in the dynamic motion. The ionized SDM of the copolymers enhanced wettability. Therefore, the advancing contact angle of the surface decreased by 45 degrees above pH 7.0, whereas wettability at low pH values was low due to the hydrophobic surface. Elution behavior of the selected four model proteins with different pI (bovine serum albumin (pI 5.0), insulin (pI 5.3), fibrinogen (pI 6.1) and myoglobin (pI 7.1)) was investigated by using pH-sensitive copolymer-modified glass beads at four pH values (5.0, 6.0, 7.0 and 8.0). Retention time of the proteins in pH-sensitive aqueous chromatography was easily regulated by eluent pH that controls the proportion of electrostatic and hydrophobic interactions between the stationary phase and the protein. At pH 5.0, hydrophobic interaction on the deionized polymer-grafted surface is an important force in separating the proteins. However, identification of the proteins at pH 7.0 was effectively facilitated due to the electrostatic interaction between the negatively charged polymer support and positive charges on protein.