Capillary electrochromatography of proteins on an anion exchanger column
Capillary electrochromatography of proteins on an anion exchanger column
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DOI：
10.1021/ac000114t
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发表时间：
2000-07-15
影响因子：
7.4
通讯作者：
Horváth, C
中科院分区：
文献类型：
--
作者：
Zhang, J;Huang, X;Horváth, C
使用50 μ m-i. d.填充有5 μ m二氧化硅珠粒的熔融二氧化硅毛细管，所述二氧化硅珠粒具有强阴离子交换剂功能，在色谱表面处连接到亲水间隔物。首先用异双官能硅烷化剂处理硅质微球和毛细管内壁，随后与含有季铵基团的乙烯基单体反应以形成“触手”阴离子交换剂。牛碳酸酐酶，α-乳白蛋白，大豆胰蛋白酶抑制剂，和卵清蛋白的混合物分离使用CEC的等度洗脱在同向模式与磷酸盐缓冲液，pH值为7.0，含有氯化钠。通过研究盐浓度对蛋白质分离的影响，阐明了等度CEC在阴离子交换柱上对蛋白质的保留机理。CEC的蛋白质分离的潜力与高分辨率也证明了伴清蛋白和血红蛋白变体的电色谱图。结果揭示了等度CEC分离蛋白质的机理，认为其是静电作用的色谱保留和电泳迁移的结合。假设这两种机制对总迁移速度的贡献是相加的，推导出电色谱分离方程，并与HPLC中的分离方程进行比较，以揭示CEC相对于HPLC中的分离度提高的成分。通过比较CEC在等度平台上的峰容量与用等度和梯度洗脱HPLC获得的峰容量，还检查了CEC的优势。
Capillary electrochromatography (CEC) of proteins was carried out using 50-mu m-i.d. fused-silica capillaries packed with 5-mu m silica beads having strong anion-exchanger functions attached to hydrophilic spacers at the chromatographic surface. The siliceous microspheres and the capillary innerwall were treated first with a heterobifunctional silanizing agent and reacted subsequently with a vinyl monomer containing quaternary ammonium groups to form a "tentacular" anion exchanger. A mixture of bovine carbonic anhydrase, alpha-lactalbumin, soybean trypsin inhibitor, and ovalbumin was separated using CEC by isocratic elution in the codirectional mode with aqueous phosphate buffer, pH 7.0, containing sodium chloride. The retention mechanism of isocratic CEC for proteins on the anion-exchanger column was illustrated by the results of a study on the effect of salt concentration on the separation. The potential of CEC for protein separation with high resolution was also demonstrated by electrochromatograms of conalbumin and hemoglobin variants. The results shed light on the mechanism of protein separation by isocratic CEC, which is believed to be a combination of chromatographic retention by electrostatic interactions and electrophoretic migration. Assuming that the contributions of the two mechanisms to the overall migration velocity are additive, an electrochromatographic resolution equation was derived and compared to the resolution equation in HPLC to reveal the constituents responsible for the enhancement of resolution by CEC with respect to that in HPLC. The advantage of CEC was also examined by comparing peak capacities in CEC on an isocratic platform with peak capacities obtained with isocratic and gradient elution HPLC.