We have prepared various poly(N-isopropylacrylamide) (PIPAAm)-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) by changing graft densities and brush chain lengths. The prepared surfaces were characterized by chromatographic analysis using the modified silica beads as chromatographic stationary phases. ATRP initiator (2-(m,p-chloromethylphenyl)etliyltrichlorosilane) density on silica bead surfaces was modulated by changing the feed composition of the self-assembled monolayers (SAMs) of mixed silane coupling agents consisting of ATRP initiator and phenethyltrichlorosilane on the surfaces. IPAAm was then polymerized on SAM-modified silica bead surfaces by ATRP in 2-propanol at 25 degrees C. The chain length of the grafted PIPAAm was controlled by simply changing the ATRP reaction time at constant catalyst concentration. The thermoresponsive surface properties of the PIPAAm-grafted silica beads were investigated by ternperature-dependent elution behavior of hydrophobic steroids from the Surfaces using Milli-Q water as a mobile phase. On the surfaces grafted with shorter PIPAAm chains, longer retention times for steroids were observed on sparsely grafted PIPAAm surfaces compared to dense PIPAAm brushes at low temperature, because of hydrophobic interactions between the exposed phenethyl groups of SAMs on silica surfaces and steroid molecules. Retention times for steroids on dilute PIPAAm chain columns decreased with temperature similarly to conventional reverse-phase chromatographic modes on octadecyl columns. This effect was due to limited interaction of Solutes with the PIPAAm-grafted Surfaces. Retention times for steroids on dilute PIPAAm brush surfaces with longer PIPAAm chains became greater above the PIPAAm transition temperature. At low-temperature regions, hydrated and expanded PIPAArn at low temperatures prevented hydrophobic interactions between the phenethyl group of SAMs on the silica bead surfaces and steroid molecules. Retention times for steroids on a dense PIPAArn brush column increased with temperature since solvated polymer segments within the dense brush layer undergo dehydration over a broad range of temperatures. In conclusion, PIPAArn graft density has a crucial influence on the elution behavior of steroids because of the interaction of analytes with silica bead interfaces, and because of the characteristic dehydration of PIPAArn in dense-pack brush surfaces.