Yang Yuanzhong, Boysen Reinhard I, Chen Jenny I-Chen, Keah Hooi Hong, Hearn Milton T W
Australian Research Council Special Research Centre for Green Chemistry, Centre for Bioprocess Technology, Australian Centre for Research on Separation Science, Monash University, PO Box 23, Clayton, Victoria 3800, Australia.
J Chromatogr A. 2003 Aug 15;1009(1-2):3-14. doi: 10.1016/s0021-9673(03)00446-1.
The separation of two different sets of synthetic peptides has been investigated by high-performance capillary zone electrophoresis utilising naked, fused silica capillaries. The effects of electrolyte pH, buffer concentration, capillary length and electric field strength on the separation efficiency and selectivity were systematically varied, with the highest resolution achieved with buffer electrolytes of low pH and relatively high ionic strength. Under optimised separation conditions utilising the "short end injection" separation approach with negative electric field polarity, a series of eight structurally-related synthetic peptides were baseline resolved within 4 min without addition of any modifier of the background electrolyte with separation efficiencies in the vicinity of 600000 theoretical plates/m. Further significant enhancement of separation efficiencies could be achieved by taking advantage of the "long end injection" approach with positive electric field polarity. The outcome of these experimental variations parallels the "sweeping" effect that has been observed in the capillary electrochromatographic and micellar electrokinetic separations of polar molecules and permits rapid resolution of peptides with focusing effects. In addition, small changes in the electrolyte buffer pH and concentration were found to have a significant impact on the selectivity of synthetic peptides of similar intrinsic charge. These observations indicate that multi-modal separation mechanisms operated under these conditions with the unmodified fused silica capillaries. This study, moreover, documents additional examples of peptide-specific multi-zoning behaviour in the high-performance capillary zone electrophoretic separation of synthetic peptides.
利用裸熔融石英毛细管，通过高效毛细管区带电泳研究了两组不同合成肽的分离。系统地改变了电解质pH值、缓冲液浓度、毛细管长度和电场强度对分离效率和选择性的影响，在低pH值和相对高离子强度的缓冲电解质条件下实现了最高分辨率。在优化的分离条件下，采用“短端进样”分离方法和负电场极性，在不添加任何背景电解质改性剂的情况下，一系列八个结构相关的合成肽在4分钟内实现基线分离，分离效率接近600000理论塔板数/米。通过采用“长端进样”方法和正电场极性，可以进一步显著提高分离效率。这些实验变化的结果与在毛细管电色谱和胶束电动分离极性分子中观察到的“扫集”效应相似，并允许通过聚焦效应快速分离肽。此外，发现电解质缓冲液pH值和浓度的微小变化对具有相似固有电荷的合成肽的选择性有显著影响。这些观察结果表明，在这些条件下，未改性的熔融石英毛细管存在多模式分离机制。此外，本研究记录了在合成肽的高效毛细管区带电泳分离中肽特异性多区域行为的其他实例。
