Effect of Water Content in Mobile Phase on Chromatographic Behaviors of Low Molecular Compounds in RP-HPLC using Methacrylate-based Monolithic Column

Abstract

The effect of water content in a mobile phase on chromatographic behavoirs was studied with a monolithc stationary phase of poly(butyl methacrylate-co-ethylene dimethacrylate). A drastic decrease in the separation efficiency was observed when the water content was up to 90%, i.e., 10% acetonitrile (ACN) aqueous solution. Interestingly, the magnitude of the efficiency decrease significantly depended on the retention factor of the analytes, in particular, the separation efficiencies of the slightly retained compounds (0.1 < k < 1, in 10% ACN) were inferior. Furthermore, with a mobile phase of 10% ACN, the acceleration of the flow rate decreased the retention factor of these less retained compounds. The higher flow rate suppressed the interaction in RP-HPLC only when the water rich solution was used. The reduction in the retention factor produced non-theoretical relations in an H-u plot. The change in the monolith structure was also indicated by the variation in the permeability in 10% ACN.