On-chip capillary electrophoresis with uncoated and polyvinyl alcohol-coated glass channels in aqueous and nonaqueous dimethyl sulfoxide (DMSO) background electrolyte (BGE) solutions was applied in the separation of five amines derivatized with fluorescein-5-isothiocyanate. In aqueous BGE at pH 9.2, baseline separation of the analytes was not achieved on uncoated glass chips, but the separation was clearly improved when the chip channels were coated with polyvinyl alcohol (PVA). Separation was successful in nonaqueous DMSO electrolyte solution containing ammonium acetate and sodium methoxide, on both uncoated and PVA-coated glass microchips. The differences in the pK(a) values of analytes were probably amplified in DMSO, and all five analytes were at least partly dissociated and were separated. Because the viscosity of DMSO is higher than that of water, the migration times were longer in DMSO.