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微量制备毛细管区带电泳和毛细管等电聚焦中的馏分收集

Fraction collection in micropreparative capillary zone electrophoresis and capillary isoelectric focusing.

作者信息

Minarik M, Foret F, Karger B L

机构信息

Barnett Institute and Department of Chemistry, Northeastern University, Boston, MA 02115, USA.

出版信息

Electrophoresis. 2000 Jan;21(1):247-54. doi: 10.1002/(SICI)1522-2683(20000101)21:1<247::AID-ELPS247>3.0.CO;2-E.

Abstract

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 microm inner diameter (ID) were employed in CZE and 100 microm 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).

摘要

本文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。收集步骤的精确计时基于测定毛细管末端附近两个检测点之间每个单独区带的速度。在收集前不久测定区带速度,消除了对整个柱中恒定分析物速度的需求。因此,具有大进样体积的CZE中的样品堆积以及CIEF中的区带聚焦都可以以高收集精度加以利用。在微制备模式下,CZE采用内径为200微米的毛细管,CIEF采用内径为100微米的毛细管。使用鞘流馏分收集器在收集过程中维持恒定电流。通过用半透膜封闭入口处的分离系统,抑制了由于虹吸作用产生的大量液体流动以及鞘流液滴压力引起的回流。在CZE模式下,通过从荧光衍生化的寡糖梯中分离出各个峰来证明馏分收集器的性能。在CIEF模式下,展示了从标准混合物中收集几种蛋白质,随后通过基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF-MS)对每个蛋白质馏分进行后续分析。

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