In this study, electrically driven separations of charged biomolecules including ribonucleotides and peptides were investigated using various surface modified columns. These columns included cationic, neutral and anionic polymeric-coated columns. Experiments demonstrated that for neutral polymer-coated columns, solutes eluted according to their electrophoretic mobilities. Using surface charged columns, the elution of charged solutes was determined by their electrophoretic mobilities, electroosmotic flow rates and interactions between charged solutes and the charged column inner wall. Retention factors were used to investigate the interactions between solutes and the column inner wall. Column efficiency measurements for charged solutes on charged surface columns were also examined, and the results were compared with those obtained using neutral polymer-coated columns. It was found that separations of multivalent biomolecules were difficult using charged surface columns because of either strong adsorption or high electrophoretic mobilities.