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We recently reported the new concept of temperature-responsive liquid chromatography using temperature-responsive poly(N-isopropylacrylamide)-modified surfaces as high-performance liquid chromatography media with aqueous mobile phases. Incorporation of hydrophobic sites is an important factor to improve the efficacy (selectivity and retention) of temperature-responsive chromatography. Toward this goal, we have synthesized semitelechelic copolymers of N-isopropylacrylamide (IP-AAm) and butyl methacrylate (BMA) having reactive terminal functional groups using telomerization. The lower critical solution temperatures of the copolymers shift to lower temperatures with increasing hydrophobic BMA content in the poly(IPAAm-co-BMA) relative to that of the IPAAm homopolymer. This temperature-responsive semitelechelic copolymer was grafted to the surface of (aminopropyl)silica through the reaction of activated ester-amine coupling. The polymer-modified silica was used as a column packing material. Separation of a mixture of five steroids having various hydrophobicities was investigated. Retention of steroids on poly(IPAAm-co-BMA)-modified columns is increased with an increase in column temperature. The capacity factors for steroids on the copolymer-modified silica beads was much larger than that on homopolymer PIPAAm-modified columns. The capacity factor for testosterone at 50 [degrees] C was 33.8 for poly(IPAAm-co-BMA) containing 5 mol % BMA, while that for the PIPAAm homopolymer was 15.0 at the same temperature. The influence of column temperature on steroid retention behavior on copolymer-modified stationary phases was significant compared with the case of homopolymer-modified columns. Furthermore, retention times for steroids increased remarkably with increasing BMA composition. The temperature-responsive elution behavior for the steroids was strongly affected by the hydrophobicity of the grafted polymer chains on silica surfaces. Possible protein separation in temperature-responsive liquid chromatography was explored using insulin chains A and B, [Beta]-endorphin fragment 1-27. On IBc-3.2-modified silica column, these three peptides were successfully separated at 30 [degrees] C with 0.5 M NaCl aqueous solution (pH 2.1) as mobile phase. The retention times of these peptides were related to the number of hydrophobic amino acid residues in the peptides. In the proposed chromatography system, elution of target substances is controlled only by a small change in column temperature without any further modification of the aqueous mobile phase.

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 IP-AAm 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.

Recently, green chemistry has become one of the most important subjects of science for environmental pollution prevention. Here, we report development of a novel chromatographic technology for phenylthiohydantoin (PTH)-amino acid analyses in which only aqueous solution is used as the mobile phase. We have devised HPLC adsorbents (stationary phase) by modifying the surfaces of microparticulate silica gel using functional polymers. The thermoresponsive copolymer, poly(N-isopropylacrylamide-co-n-butyl methacrylate) (IBc) was used to modify the silica stationary phase surfaces. This polymer-grafted surface exhibits temperature-regulated hydrophilic/hydrophobic property changes in water. PTH-amino acid interactions with this surface are readily modulated by changing the column temperature using an isocratic aqueous mobile phase. Increasing hydrophobic interactions between more hydrophobic PTH-amino acids with hydrophobized polymer-grafted surfaces at elevated mobile phase temperatures is used for the effective separation of PTH-amino acids in aqueous solution. This study is aimed at the development of novel separation processes, which are also environmentally benign, for use with biochemical substances in order to meet the growing needs of the life sciences and biotechnology. The method is useful for various separations in life science so that proteins can maintain their biological activity and enzymes, their enzymatic activity.