Temperature-Responsive Chromatography Using Poly(N-isopropylacrylamide)-Modified Silica

A new concept in chromatography is proposed that utilizes a temperature-responsive surface with a constant aqueous mobile phase. The surface of the silica stationary phase in high-performance liquid chromatography (HPLC) has been modified with temperature-responsive polymers to exhibit temperature-controlled hydrophilic/hydrophobic changes. Poly(N-isopropylacrylamide) (PIPAAm) was grafted onto (aminopropyl)silica using an activated ester-amine coupling method. These grafted silica surfaces show hydrophilic properties at lower temperatures which, as temperature increases, transform to hydrophobic surface properties. The elution profile of five mixed steroids on an HPLC column packed with this material depends largely on the temperature of the aqueous mobile phase. Retention times increase with increasing temperature without any change in the eluent. Changes in the retention times of hydrophobic steroids were larger than those for hydrophilic steroids. The temperature-responsive interaction between PIPAAm-modified silica and these steroids is proposed to result from changes in the surface properties of the HPLC stationary phase by the transition of hydrophilic/hydrophobic surface-grafted IPAAm polymers. We demonstrate a novel and useful new chromatography system in which surface properties and the resulting function of the HPLC stationary phase are controlled by external temperature changes. This method should be effective in biological and biomedical separations of peptides and proteins using only aqueous mobile phases.