Hydrophobized thermoresponsive polymer brush on porous monolithic silica for effective bioseparations

摘要

Introduction: Thermoresponsive chromatography using poly(N-isopropylacrylamide)(PIPAAm) grafted silica beads have been investigated as new types of chromatography whose stationary phase property can be controlled simply by changing temperature.For developing more efficient thermoresponsive chromatography system, we have prepared hydrophobized thermoresponsive copolymer modified monolithic silica column, and investigated their properties as chromatographic materials. Materials and Methods: ((Chloromethyl)phenylethyl)trimethoxysilane (CPTMS), ATRP-initiator, was immobilized on monolithic silica surfaces by flowing a toluene solution containing ATRP-initiator into monolithic silica columns using a HPLC pump. Copolymerization of IPAAm and n-butyl methacrylate (BMA) were performed onto monolithic-silica-rod surfaces through surface-initiated ATRP using CuCl/CuCl_2/Me_6TREN catalyst and 2-propanol as a reaction solution at 25 °C for 4 and 16 h (Fig.1). The prepared copolymer brush modified monolithic silica column was connected to an HPLC system, and the elution behaviour of benzoic acids and insulin fragments were observed at various temperatures. For the comparison, same copolymer brush modified silica beads packed column was prepared. Results and Discussion: CHN elemental analysis and GPC measurement revealed that grafted amount and grafted density of P(IPMm-co-BMA) a on monolithic silica were 5.87 mg/m~2 and 0.116 chainsm~2, respectively. These indicated that large amount of copolymer was grafted on monolithic silica surface. Fig.2 shows temperature dependent elution profiles of benzoic acids and phenol on the prepared column. Separation was performed at high temperature, because hydrophobic interaction between analytes and grafted copolymer enhanced at high temperature due to the dehydration of the copolymer. Monolithic silica column exhibit high-resolution separation of benzoic acids group compared to beads packed column. Fig.3 shows elution profile of insulin fragments. Monolithic silica columns performed baseline separation of insulin fragment. These results indicated that thermoresponsive hydrophobic monolithic silica column would be useful for bioseparation application. Conclusions: Hydrophobized thermoresponsive copolymer brush modified porous monolithic silica would be useful for effective bioseparation simply by changing column temperature.