We have been investigating a new concept of chromatography, an environmental-responsive chromatography, using these functional polymers modified packing materials for HPLC. We designed and synthesized thermo-responsive copolymers with N-isopropylacrylamide (NIPAAm), butyl methacrylate (BMA) and other comonomers. These polymers underwent a reversible phase transition from water-soluble forms into aggregates by changing the temperature. The copolymers were grafted onto aminopropyl silica beads, and the products were evaluated as HPLC packing materials. In a chromatographic system using the thermo-responsive polymers, we could separate steroids, amino acids and proteins with a variety of hydrophobicities using a sole aqueous mobile phase. The retention on the polymer-modified stationary phase remarkably changed upon changing the temperature. With increasing temperature, an increased interaction between analytes and polymer-grafted surfaces of the stationary phases was observed. The drastic and reversible surface hydrophilic-hydrophobic property alteration should be due to rapid changes in the polymer hydration state around the polymer's transition temperature. A solvent gradient elution-like effect could be achieved with a single mobile phase by programmed temperature changes during chromatographic runs. An environmental-responsive analytical system using a functional polymer-modified surface would be highly useful to control the function and property of the stationary phase for HPLC only by changing the temperature with an aqueous solvent. These systems have advantages over conventional HPLC, including improved maintenance of recovered solute bioactivity and the elimination of organic solvent use.
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