Olvecká Eva, Kaniansky Dusan, Pollák Branislav, Stanislawski Bernd
Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
Electrophoresis. 2004 Nov;25(21-22):3865-74. doi: 10.1002/elps.200406080.
This feasibility study deals with the separations of proteins by an on-line combination of zone electrophoresis (ZE) with isotachophoresis (ITP) on a poly(methylmethacrylate) column-coupling (CC) chip with integrated conductivity detection. ITP and ZE provided specific analytical functions while performing the cationic mode of the separation. ITP served, mainly, for concentrations of proteins and its concentrating power was beneficial in reaching a low dispersion transfer (injection) of the proteinous constituents, loaded on the CC chip in a 960 nL volume, into the ZE separation stage. This was complemented by an electrophoretically driven removal of the sample constituents migrating in front of the focused proteins from the separation system before the ZE separation. On the other hand, ZE served as a final separation (destacking) method and it was used under the separating conditions providing the resolutions and sensitive conductivity detections of the test proteins. In this way, ITP and ZE cooperatively contributed to low- or sub-microg/mL concentration detectabilities of proteins and their quantitations at 1-5 microg/mL concentrations. However, a full benefit in concentration detectabilities of proteins, expected from the use of the ITP-ZE combination, was not reached in this work. Small adsorption losses of proteins and detection disturbances in the ZE stage of separation, very likely due to trace constituents concentrated by ITP, appear to set limits in the detection of proteins in our experiments. The ITP-ZE separations were carried out in a hydrodynamically closed separation compartment of the chip with suppressed hydrodynamic and electroosmotic flows of the electrolyte solutions. Such transport conditions, minimizing fluctuations of the migration velocities of the separated constituents, undoubtedly contributed to highly reproducible migrations of the separated proteins (fluctuations of the migration time of a particular protein were typically 0.5% RSD in repeated ITP-ZE runs).
本可行性研究涉及在具有集成电导检测功能的聚甲基丙烯酸甲酯柱耦合（CC）芯片上，通过区带电泳（ZE）与等速电泳（ITP）的在线联用实现蛋白质分离。在进行阳离子模式分离时，ITP和ZE发挥了特定的分析功能。ITP主要用于蛋白质浓缩，其浓缩能力有助于将以960 nL体积加载到CC芯片上的蛋白质成分低分散转移（进样）到ZE分离阶段。在ZE分离之前，通过电泳驱动从分离系统中去除在聚焦蛋白质之前迁移的样品成分，对这一过程起到了补充作用。另一方面，ZE用作最终分离（去堆积）方法，在提供测试蛋白质分辨率和灵敏电导检测的分离条件下使用。通过这种方式，ITP和ZE共同实现了低至亚微克/毫升浓度的蛋白质检测能力及其在1 - 5微克/毫升浓度下的定量分析。然而，在本研究中，未实现使用ITP-ZE联用预期的蛋白质浓度检测的全部优势。蛋白质的少量吸附损失以及ZE分离阶段的检测干扰，很可能是由于ITP浓缩的痕量成分所致，这似乎在我们的实验中对蛋白质检测设置了限制。ITP-ZE分离在芯片的流体动力学封闭分离隔室中进行，抑制了电解质溶液的流体动力学和电渗流。这种传输条件使分离成分的迁移速度波动最小化，无疑有助于分离蛋白质的高度可重复迁移（在重复的ITP-ZE运行中，特定蛋白质迁移时间的波动通常为0.5%相对标准偏差）。
