Chen Jian-Lian
School of Pharmacy, China Medical University, Taichung 40402, Taiwan.
J Chromatogr A. 2009 Aug 21;1216(34):6236-44. doi: 10.1016/j.chroma.2009.07.017. Epub 2009 Jul 16.
Two open-tubular (OT) capillary electrochromatographic (CEC) columns were prepared by chemically bonding ionizable mono-(2-(methacryloyloxy)ethyl) succinate (MES) and phthalate-functionalized (MEP) ligands onto silica hydride-based phases through surface etching, silanization, and hydrosilation reactions, starting with a bare fused-silica tube. An analysis of the effect of performance of electrophoretic flow (EOF) on the changes in pH values, ionic strength, and the amount of acetonitrile modifiers helped to reveal that some silanol groups remained in the surface composite of the modified capillaries and to prove that MEP capillaries actually exerted greater EOF than MES ones. To explore the potential utilization of these two columns in various fields, three categories of samples, which spanned a wide range of polarities, were prepared and analyzed through many systematic trials of optimizing CEC conditions. For the separation of a mixture of nucleosides and thymine, guanine and adenine with purine uncleobases, which exhibit greater aromaticity than pyrimidine nucleobases, performed a higher retention in the MEP capillary through a pi-pi interaction than in the MES capillary. While four steroids were used as test samples, their migration order revealed that the MES stationary phase is hydrophilic in comparison with the MEP. An addition of methanol modifier (30%, v/v) into 10 mM borate buffer (pH 9.55 for MEP; pH 10.0 for MES) was necessary to accomplish a baseline separation of nine flavonoids in the MEP and MES capillaries. Studies on the elution order of these solutes revealed the presence of chromatographic activity in addition to electrophoretic migration. Especially in the MEP capillary, hydrophobic characteristics and pi-pi interactions with aromatic solutes were found and further improved to resolve an enantiomeric pair, catechin and epicatechin. Overall, the hydride-based stationary phases with ionizable ligands were successfully applied to the OT-CEC separations, and these results confidently propose an ideal route to the synthesis of a novel OT-CEC column.
以裸露的熔融石英管为起始材料，通过表面蚀刻、硅烷化和硅氢化反应，将可电离的单（2 - （甲基丙烯酰氧基）乙基）琥珀酸酯（MES）和邻苯二甲酸酯官能化（MEP）配体化学键合到氢化硅基固定相上，制备了两根开管（OT）毛细管电色谱（CEC）柱。对电泳流（EOF）性能随pH值、离子强度和乙腈改性剂用量变化的分析，有助于揭示改性毛细管表面复合物中仍存在一些硅醇基团，并证明MEP毛细管的EOF实际上比MES毛细管的更大。为了探索这两根柱在各个领域的潜在应用，制备了三类极性范围广泛的样品，并通过许多优化CEC条件的系统试验进行了分析。对于核苷混合物以及胸腺嘧啶、鸟嘌呤和腺嘌呤与嘌呤无环碱基的分离，与嘧啶核苷碱基相比具有更大芳香性的嘌呤无环碱基，通过π-π相互作用在MEP毛细管中的保留率高于MES毛细管。当使用四种甾体作为测试样品时，它们的迁移顺序表明MES固定相与MEP相比具有亲水性。向10 mM硼酸盐缓冲液（MEP为pH 9.55；MES为pH 10.0）中添加甲醇改性剂（30%，v/v）对于在MEP和MES毛细管中实现九种黄酮类化合物的基线分离是必要的。对这些溶质洗脱顺序的研究揭示了除电泳迁移外还存在色谱活性。特别是在MEP毛细管中，发现了与芳香族溶质的疏水特性和π-π相互作用，并进一步优化以分离对映体对儿茶素和表儿茶素。总体而言，带有可电离配体的氢化硅基固定相成功应用于OT-CEC分离，这些结果为新型OT-CEC柱的合成提供了一条理想途径。
