Rapid separation of nucleosides by capillary electrochromatography with a methacrylate-based monolithic stationary phase

Separation of nucleosides by capillary electrochromatography with a methacrylate-based monolithic column is described. Because the nucleosides are relatively hydrophilic compounds, chromatographic partition is very weak on non-polar stationory phases. A small quantity of organic modifier is usually required to increase their retention factors but, because the wettability of the stationary phase by the mobile phase is rather poor, bubble formation can be a significant problem. This situation was improved by use of a polymeric monolithic column with higher level charged monomer in the polymerization mixture, because of the presence of charged moieties on the surface of the monolithic stationary phase. The very high stability under conditions of very low organic modifier concentration used in the experiment showed that the wettability of the stationary phase by the mobile phase was satisfactory The effect of pH, organic modifier content, and applied voltage on the separation was investigated. Baseline separation of six nucleosides was achieved within 4.5 min on this type of column. Because of the presence of charged moieties and butyl groups on the surface of the monolithic column, the mechanism of separation might involve both ion-exchange and hydrophobic interactions.