We have prepared poly( N-isopropylacrylamide) (PIPAAm) brush-grafted surfaces with varied temperature-responsive hydrophobic properties through surface-initiated atom transfer radical polymerization (ATRP). These temperature-responsive surfaces were characterized by chromatographic analysis using modified silica beads as a chromatographic stationary phase in aqueous mobile phase. Mixed silane self-assembled monolayers (SAMs) comprising ATRP initiator and silanes with various terminal functional groups were formed on the silica bead surfaces. IPAAm was then polymerized by ATRP using the CuCl/CuCl2/Me6TREN catalyst system in 2-propanol at 25 degrees C for 16 h. The chromatographic retention behavior of steroids on the resulting PIPAAm brushes made on more polar silane components was distinct from that on more apolar silane interfaces. Retention times for steroids on PIPAAm mixed apolar silane graft interfaces were significantly longer than those on analogous polar silane interfaces due to enhanced dehydration of PIPAAm brushes on apolar silane-grafted surfaces. Changes in retention factor, k', on polar silane PIPAAm-grafted interfaces were relatively large compared to that on apolar PIPAAm grafted interfaces due to larger hydration/dehydration alterations of grafted PIPAAm brushes on the former surfaces. Applied step-temperature gradients from 50 to 10 degrees C show that PIPAAm brushes on polar silane interfaces tend to hydrate more, leading to shorter retention times. In conclusion, the polarity of the grafted interface significantly influences the grafted PIPAAm brush hydration/dehydration characteristics and subsequently also the temperature-modulated separation of bioactive compounds in all-aqueous chromatography.