We developed a new method for high performance liquid chromatography (HPLC) using packing materials modified with a temperature responsive polymer, poly(N-isopropylacrylamide) (PNIPAAm). A homogeneous PNIPAAm polymer and its copolymer with butyl methacrylate (BMA) were synthesized and grafted to aminopropyl silica by activated ester-amine coupling and they were used as packing materials. These grafted silica surfaces show hydrophilic properties at lower temperatures and, as temperature increases, transform to hydrophobic surface properties. These features are due to rapid changes in the polymer conformation that is attributed to the mobility of grafted polymers. Using the column packed with PIPAAm-modified silica, separation of steroids was carried out by changing temperature. With increasing temperature, the interaction between solutes and PNIPAAm-grafted surfaces of the stationary phases increased. The temperature-dependent resolution of steroids was achieved using only water as a mobile phase. The separation selectivity and retention are controlled by a small change in column temperatures without any change in the eluent. The separation of the mixture of three peptides, insulin chains A and B and a β-endorphin fragment 1-27, was achieved by changing column temperature with a NaCl aqueous solution as a sole eluent. A gradient elution-like effect in HPLC is demonstrated with a single mobile phase by controlling external temperature. The retention times of peptides and proteins were controlled both by column temperatures and by NaCl concentration in the aqueous mobile Phases in this chromatographic system.
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