Ikegami Tohru, Horie Kanta, Jaafar Jafariah, Hosoya Ken, Tanaka Nobuo
Department of Biomolecular Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, 606-8585, Japan.
J Biochem Biophys Methods. 2007 Feb 23;70(1):31-7. doi: 10.1016/j.jbbm.2006.09.010. Epub 2006 Oct 5.
Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u=4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u=8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.
通过在经N-(3-三乙氧基甲硅烷基丙基)甲基丙烯酰胺锚定基团改性的整体硅胶毛细管柱上进行丙烯酸的柱上聚合，制备了弱阳离子交换(WCX)和亲水作用色谱(HILIC)模式柱。聚合物涂层柱可用于HILIC模式下吡啶基氨基(PA)-糖和肽的分离，包括牛血清白蛋白的胰蛋白酶消化产物，而在弱阳离子交换模式下，即使在高线速度下也可用于蛋白质和核苷的分离。与以相同方式制备的聚丙烯酰胺涂层整体硅胶毛细管柱相比，聚(丙烯酸)涂层整体硅胶毛细管柱对PA-糖的保留能力更强。使用同一根柱子在pH 6.9时可有效分离蛋白质和核苷。在聚合物涂层步骤后，该柱具有良好的渗透性。结果表明，在u = 4.66 mm/s时可以高效地对蛋白质进行高速分离，而在u = 8.67 mm/s时使用该柱在一分钟内即可实现核苷的高速分离，这表明该柱将适用于多维高效液相色谱系统的二维分离。
