Mobile phase effects in reversed-phase and hydrophilic interaction liquid chromatography revisited.

Correct adjustment of the mobile phase is equally important as the selection of the appropriate column for the separation of polar compounds in LC. Both solvophobic and selective polar interactions control the retention in the Reversed Phase and Hydrophilic Interaction modes. The retention models describing the effects of the volume fraction of the strong eluent component in binary mobile phases on the sample retention factors apply in a limited mobile phase composition range. We introduced a three-parameter retention model, which provides improved prediction of retention over a broad mobile phase range, under isocratic and gradient elution conditions. The model does not imply any assumptions concerning either adsorption or partition distribution mechanism, but allows estimating retention in pure strong and in pure weak mobile phase components. The experimental retention data for phenolic acids and flavones on several core-shell columns with different types of stationary phases agree with the theory. Many polar columns with important structural hydrophobic moieties show dual retention mechanism, (Reversed Phase in water rich mobile phases and Hydrophilic Interaction at high acetonitrile concentrations). It is possible to select the mobile phase compositions in each of the two modes for separations of samples containing compounds largely differing in polarity. The three-parameter model describes the retention in each mode, with separately determined best-fit parameters. We applied the two-mode model to the retention data of sulfonamides and benzoic acid related compounds on a new polymethacrylate zwitterionic monolithic micro-column.