(475k) Preparation of Chromatography Matrices Having Thermoresponsive Polymer Brush Structure

We prepared silica stationary-phase surfaces densely grafted with Poly(N-isopropylacrylamide)(PIPAAm) by ATRP (Atom transfer radical polymerization) to investigate temperature dependent elution behavior of bioactive compounds in aqueous mobile phase. Silica beads were cleaned in hydrochloric acid, and 1-trichlorosil-2-(m,p-chloromethylphenyl) ethane was immobilized to them. The reaction solution, which was prepared by dissolving N-isopropylacrylamide, CuCl and Me₆TREN into degassed DMF, was poured into the silica beads placed glass vessel. The ATRP reaction proceeded for predetermined reaction period at 25C under continuous shaking. The PIPAAm grafted silica beads was characterized by the elemental analysis and SEM observation. Temperature dependent elution behavior of bioactive compounds in aqueous mobile phase was investigated with an HPLC system. The amount of grafted PIPAAm was approximately ten times larger than that of the polymer hydrogel modified silica beads prepared by conventional polymerization method. SEM observation suggested that the polymer modification by ATRP enables uniformly coating on silica beads with large amount of PIPAAm layer. The retention behavior of steroids suggests that the amount of PIPAAm grafted onto silica beads surface should be controlled by reaction time for a separation with high resolution peak. The retention time of steroids is longer than the previously developed PIPAAm modified silica beads, and this suggests that the hydrophobicity of the surface is stronger than previously developed PIPAAm modified surfaces owing to the large amount of grafted PIPAAm onto the silica beads. Mixture solution of two peptides, insulin chain A and insulin, was applied to the densely grafted PIPAAm surfaces with changing temperature using a neutral (pH 7.0) aqueous mobile phase. The two peptides were well-resolved at 40C without introducing hydrophobic modifier and lowering pH of mobile phase. This suggests that the densely grafted PIPAAm onto silica surfaces has significantly large hydrophobicity and which enables separating peptides. In conclusion, the large amount of PIPAAm is grafted densely to the silica surfaces by ATRP, and the surface enables controlling strong hydrophobic interaction with bioactive compound. Thus, the surface would be a novel effective stationary phase for thermoresponsponsive chromatography.