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Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing

机译:微制备毛细管区带电泳和毛细管等电聚焦中的馏分收集

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摘要

A new fraction collection system for capillary zone electrophoresis (CZE) and capillary isolelectric focusing (CIEF) is described. Exact timing of the collector steps was based on determining the velocity of each individual zone measured between two detection points close to the end of the capillary. Determination of the zone velocity shortly before collection overcame the need for constant analyte velocity throughout the column. Consequently, sample stacking in CZE with large injection volumes as well as zone focusing in CIEF could be utilized with high collection accuracy. Capillaries of 200 mu m inner diameter (ID) were employed in CZE and 100 mu m ID in CIEF for the micropreparative mode. A sheath flow fraction collector was used to maintain permanent electric current during the collection. The bulk liquid flow due to siphoning, as well as the backflow arising from the sheath flow droplet pressure, were suppressed by closing the separation system at the inlet with a semipermeable membrane. In the CZE mode, the performance of the fraction collector is demonstrated by isolation of individual peaks from a fluorescently derivatized oligosaccharide ladder. In the CIEF mode, collection of several proteins from a mixture of standards is shown, followed by subsequent analysis of each protein fraction by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). [References: 49]
机译:描述了一种用于毛细管区带电泳(CZE)和毛细管等电聚焦(CIEF)的新型馏分收集系统。收集器步骤的确切计时是基于确定在靠近毛细管末端的两个检测点之间测得的每个单独区域的速度。在收集之前确定区域速度很快就克服了在整个色谱柱上保持恒定分析物速度的需求。因此,可以以很高的收集精度利用大进样量的CZE中的样品堆叠以及CIEF中的区域聚焦。对于微制备模式,在CZE中使用200微米内径(ID)的毛细管,在CIEF中使用100微米内径的毛细管。鞘流分数收集器用于在收集期间保持永久电流。通过用半透膜封闭进口处的分离系统,可以抑制由于虹吸作用引起的大量液体流动,以及由鞘流液滴压力引起的回流。在CZE模式下,馏分收集器的性能通过从荧光衍生的寡糖阶梯分离各个峰来证明。在CIEF模式下,显示了从标准混合物中收集的几种蛋白质,然后通过基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)对每个蛋白质组分进行后续分析。 [参考:49]

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