Ucon-coated columns have been used to isolate nucleotides, such as ribonucleotides, deoxyribonucleotides, and pyridine nucleotides, by capillary zone electrophoresis. This neutral, hydrophilic column coating (Ucon) significantly reduced macromolecule adsorption and electroosmotic flow, which provides the maximum resolution of the nucleotides separated under moderate buffer conditions (pH about 5-6). The relative standard deviations of the nucleotides were less than 1%. Low minimum detectable concentrations (about 2-8 microM) and quantities (about 70-360 fmol) of the ribonucleotides were obtained. A qualitative and quantitative analysis of the ribonucleotides in hybridoma cell extracts shows the applicability of the developed methodology for the determination of intracellular nucleotide pools under the desirable buffer conditions.