Electrophoresis in a chromatographic column packed with porous gel has been successfully established to preparatively separate proteins and DNA. This method shows good separative performance. Since separation of small polar nucleotides and oligonucleotides is challenging to conventional reversed phase high-performance liquid chromatography (RP-HPLC), it is of significance to establish preparative electrophoresis for laboratory-scale purification of them. In this paper, milligram of model polar samples, cytidine 5'-monophosphate (CMP) and uridine 5'-monophosphate (UMP), could be completely separated by a column (0.6 cm i.d.) packed with Sepharose 6B at pH 3.6 in 120 V/cm electric field, but never be separated without electric field. The orientation and strength of electric field could affect retention, resolution, and recovery of mononucleotides. With cathode at the column outlet and anode at the column inlet, the electric field force on UMP was strong enough to counteract the buffer flow, retain UMP at the column inlet, and markedly enhance the resolution between UMP and CMP. With this electrical retention mode, the maximum sample capacity was 7.5 mg on a column of 40 × 0.6 cm i.d. (bed volume 11.4 mL), which is much more than that of a RP-HPLC column. This electrokinetic method is a potential system for the separation of small polar compounds from natural extracts.