Australian Centre for Research on Separation Science-ACROSS, School of Chemistry, University of Tasmania, Private Bag 75, Hobart 7001, Australia.
J Chromatogr A. 2013 Jan 18;1273:26-33. doi: 10.1016/j.chroma.2012.10.068. Epub 2012 Dec 10.
Rigid monolithic cryostructures were prepared in capillary format at sub-zero temperatures and used successfully in the separation of proteins by hydrophobic interaction chromatography (HIC). The polymerization mixture consisted of poly(ethyleneglycol) diacrylate (PEGDA) M(n)∼258 as the single monomer, a mixture of dioxane and water as the porogen and N,N,N',N'-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS) as the initiator system. At sub-zero temperatures, the solvent mixture used as the porogen is frozen, leading to the formation of a polymeric structure templated by the solvent crystals that are formed. The optimization of the polymerization reaction was carried out by studying the influence of different reaction parameters including the temperature of the reaction, monomer concentration and solvent, on the porous characteristics of the polymers obtained. Separations were performed in HIC mode using 3 M ammonium sulfate in 0.1 M phosphate buffer, pH 6.9 to 0.1 M phosphate buffer, pH 6.9 over a 15 min gradient. The addition of neutral nanoparticles synthesized by mini-emulsion polymerization greatly improved the separation of the protein mixture, doubling the peak capacity of the control column without nanoparticles (from 7 to 17). Although the peak capacities and resolution values achieved were lower than those reported for conventional methacrylate monolithic columns, the use of this polymerization approach allows the preparation of polymeric structures which presented a more open porous structure and consequently exhibited significantly higher permeability than conventional polymer monoliths.
刚性整体低温毛细晶体制备，并成功应用于疏水性相互作用色谱（HIC）分离蛋白质。聚合混合物由聚乙二醇二丙烯酸酯（PEGDA）M(n)∼258 作为单体，二氧六环和水的混合物作为致孔剂，N，N，N'，N'-四甲基乙二胺（TEMED）和过硫酸铵（APS）作为引发剂体系。在低温下，用作致孔剂的溶剂混合物被冻结，导致形成由形成的溶剂晶体模板化的聚合结构。通过研究不同反应参数（包括反应温度、单体浓度和溶剂）对所得聚合物的多孔特性的影响，对聚合反应进行了优化。在 HIC 模式下，使用 3 M 硫酸铵在 0.1 M 磷酸盐缓冲液，pH 6.9 至 0.1 M 磷酸盐缓冲液，pH 6.9 进行 15 分钟梯度洗脱。中性纳米粒子的加入极大地改善了蛋白质混合物的分离，将没有纳米粒子的对照柱的峰容量提高了一倍（从 7 到 17）。尽管所达到的峰容量和分辨率值低于传统甲基丙烯酸酯整体柱报道的值，但使用这种聚合方法可以制备具有更开放多孔结构的聚合结构，从而表现出比传统聚合物整体柱显著更高的渗透性。
