Design of Temperature-Responsive Surfaces and Its Application for Aqueous Chromatography

本项目旨在开发一种新的水相色谱系统，在不改变溶质结构和功能的情况下，通过外部温度的变化来控制固定相的极性，从而分离和纯化溶质。该系统与反相色谱法有很大的不同，在反相色谱法中，固定相的极性是通过在不同混合比例的水中加入有机溶剂而改变的。在该体系中，用端为羧基的温度响应型聚n -异丙基丙烯酰胺（PIPAAm）修饰了氨基化二氧化硅固定相的表面。用剩余表面官能团滴定、x射线光电子能谱和原子力显微镜等方法证实了聚合物修饰表面的成功。这些表面表现出温度响应性的润湿性变化，在低于聚合物转变温度的较低温度下为亲水性，在高于聚合物转变温度时变为疏水性。采用pipaam接枝二氧化硅填充的高效液相色谱柱，采用水相流动相，通过改变温度，研究了5种甾体激素的洗脱谱。疏水类固醇的保留时间随温度升高而增加，而洗脱液成分没有变化。疏水甾体的洗脱时间延迟是由于这些化合物与pipaam接枝表面的相互作用增强，表面性质随着温度的升高而疏水。因此，通过控制外部温度，可以用单个流动相实现梯度洗脱效果。我们进一步研究了这种温度响应色谱法用于多肽分离的可能性。具有相似氨基酸残基数的多肽在较高温度下成功分离，其中溶质与固定相的疏水相互作用是影响分离效果的主要因素。总之，在所提出的色谱中，目标物质的洗脱仅通过柱温的微小变化来控制，而无需进一步修改水流动相。少

This project aimed to develop a novel aqueous chromatography system in which the polarity of stationary phases is controlled by external temperature changes to separate and purify solutes without changing their structure and functions. This system is quite different from reversed phase chromatography in which the polarity of stationary phases is altered by the addition of organic solvents in water with various mixing ration. In the proposed system, the surface of the aminated silica stationary phase was modified with temperature-responsive poly (N-isopropylacrylamide) (PIPAAm) with a carboxyl end. The successful modification of the surfaces with polymers were confirmed by means of titration of residual surface functional groups, X-ray photoelectron spectroscopy, and atmic force microscope. These surfaces showed temperature-responsive wettability changes, being hydrophilic at lower temperature below polymer transition temperature, and become hydrophobic above the transition temperature. … More The elution profiles of five steroids were then investigated by changing temperature using high-performance liquid chromatography column packed with PIPAAm-grafted silica using aqueous mobile phase. Retention times of hydrophobic steroids increased with increasing temperature without any change in the eluent composition. Retarded elution time of hydrophobic steroids resulted from the stronger interaction of these compounds with the PIPAAm-grafted surfaces in which the surface property becomes hydrophobic with temperature increase. As a consequence, gradient elution-like effect is achieved with a single mobile phase by controlling external temperature. We further investigated the possibility of this temperature-responsive chromatography for polypeptide separation. Polypeptides with similar number of amino acid residues were successfully separated at higher temperature where hydrophobic interaction of solutes with stationary phases are primarily influenced the separation efficacy. In conclusion, elution of target substances is controlled only by a small change in column temperature without any further modification of the aqueous mobile phase in the proposed chromatography. Less