Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.
J Chromatogr A. 2010 Jun 11;1217(24):3844-54. doi: 10.1016/j.chroma.2010.04.032. Epub 2010 Apr 18.
Two different monoliths, both containing phosphoric acid functional groups and polyethylene glycol (PEG) functionalities were synthesized for cation-exchange chromatography of peptides and proteins. Phosphoric acid 2-hydroxyethyl methacrylate (PAHEMA) and bis[2-(methacryloyloxy)ethyl] phosphate (BMEP) were reacted with polyethylene glycol diacrylate (PEGDA) and polyethylene glycol acrylate (PEGA), respectively, in 75-mum i.d. UV-transparent fused-silica capillaries by photo-initiated polymerization. The hydrophobicities of the monoliths were evaluated using propyl paraben under reversed-phase conditions and synthetic peptides under ion-exchange conditions. The resulting monoliths exhibited lower hydrophobicities than strong cation-exchange monoliths previously reported using PEGDA as cross-linker. Dynamic binding capacities of 31.2 and 269mg/mL were measured for the PAHEMA-PEGDA and BMEP-PEGA monoliths, respectively. Synthetic peptides were eluted from both monoliths in 15min without addition of acetonitrile to the mobile phase. Peak capacities of 50 and 31 were measured for peptides and proteins, respectively, using a PAHEMA-PEGDA monolith. The BMEP-PEGA monolith showed negligible hydrophobicity. A peak capacity of 31 was measured for the BMEP-PEGA monolith when a 20-min salt gradient rate was used to separate proteins. The effects of functional group concentration, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run [relative standard deviation (RSD)<1.99%] and column-to-column (RSD<5.64) reproducibilities were achieved. The performance of the monoliths in ion-exchange separation of peptides and proteins was superior to other polymeric monolithic columns reported previously when organic solvents were not added to the mobile phase.
两种不同的整体柱，均含有磷酸基团和聚乙二醇（PEG）官能团，被用于合成用于肽和蛋白质的阳离子交换色谱。磷酸 2-羟乙基甲基丙烯酸酯（PAHEMA）和双[2-（甲基丙烯酰氧基）乙基]磷酸酯（BMEP）分别与聚乙二醇二丙烯酸酯（PEGDA）和聚乙二醇丙烯酸酯（PEGA）在 75-μm i.d. 的紫外透明熔硅毛细管中通过光引发聚合反应得到。通过反相条件下使用对丙基苯甲酸和离子交换条件下使用合成肽评估整体柱的疏水性。所得到的整体柱的疏水性比以前报道的使用 PEGDA 作为交联剂的强阳离子交换整体柱的疏水性低。PAHEMA-PEGDA 和 BMEP-PEGA 整体柱的动态结合容量分别为 31.2 和 269mg/mL。在没有向流动相中添加乙腈的情况下，两种整体柱都可以在 15min 内洗脱合成肽。使用 PAHEMA-PEGDA 整体柱，分别测量到 50 和 31 的肽和蛋白质的峰容量。BMEP-PEGA 整体柱的疏水性可以忽略不计。当使用 20min 的盐梯度速率分离蛋白质时，BMEP-PEGA 整体柱的峰容量为 31。研究了官能团浓度、流动相 pH 值、盐梯度速率和疏水性对分析物保留的影响。实现了良好的运行间（相对标准偏差（RSD）<1.99%）和柱间（RSD<5.64%）重现性。当流动相中不添加有机溶剂时，整体柱在肽和蛋白质的离子交换分离中的性能优于以前报道的其他聚合物整体柱。
