Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.