The effect of the conformation of a polymeric pseudostationary phase on performance and selectivity in electrokinetic chromatography was studied using an amphiphilic pH-responsive polymer that forms compact intramolecular aggregates (unimer micelles) at low pH and a more open conformation at high pH. The change in conformation was found to affect the electrophoretic mobility, retention, selectivity, and separation efficiency. The low-pH conformer has higher electrophoretic mobility and greater affinity for most solutes. The unimer micelle conformation was also found to provide a solvation environment more like that of micelles and other amphiphilic self-associative polymers studied previously. It was not possible to fully characterize the effect of conformation on efficiency, but very hydrophobic solutes with long alkyl chains appeared to migrate with better efficiency when the unimer micelle conformation was employed. The results imply that polymers with a carefully optimized lipophilic-hydrophilic balance that allow self-association will perform better as pseuclostationary phases. In addition, the results show that electrokinetic chromatography is a useful method for determining the changes in solvation environment provided by stimuli-responsive polymers with changes in the conditions.