Zirconiananoparticles-coated column for the capillary electrochromatographic separation of iron-binding- and phosphorylated-proteins

Abstract

A ZrO₂ nanoparticles (ZrO₂NPs)-coated column was prepared through a sol–gel process using zirconium(IV) oxychloride, which reacted with silanol groups of the fused-silica capillary. The condensation reaction was carried out at 350 °C for 8 h. Electroosmotic flow (EOF) measurements and scanning electron microscopy (SEM) images were used to characterize the ZrO₂NPs fabricated on the inner wall of the capillary. Below the pI value (pH 5–6), cathodic EOF elucidated that the phosphate buffer adsorbs tightly on the zirconia surface, resulting in a negatively charged surface. In this work, iron-binding proteins, phosphorylated proteins and glycoproteins were selected as the model compounds. The effects of pH, concentration, buffer type and the organic modifier were studied to optimize the separation efficiency. Iron-binding proteins exhibited a retention time for myoglobin (Mb) < hemoglobin (Hb), which corresponded to the binding constants for ZrO₂NPs. The α- and β-subunit of Hb could be separated in borate buffer (20 mM, pH 9.0) with MeOH (20%, v/v). Greater affinity of α-casein and bovine serum albumin (BSA) for the stationary phase as the pH decreased was found by comparison with that of conalbumin (ConA) and transferrin (Tf). Interestingly, 14 peaks for glycoisoforms of ovalbumin (OVA) were observed using borate buffer (40 mM, pH 9.0). The established method was also applied to the determination of analytes in the egg whites of chicken and duck eggs.