Ross David, Romantseva Eugenia F
National Institute of Standards and Technology 100 Bureau Drive, Gaithersburg, Maryland 20899, USA.
Anal Chem. 2009 Sep 1;81(17):7326-35. doi: 10.1021/ac901189y.
Gradient elution moving boundary electrophoresis (GEMBE) is a recently described technique for electrophoretic separations in short (1-3 cm) capillaries or microchannels. With GEMBE, the electrophoretic migration of analytes is opposed by a bulk counterflow of separation buffer through the separation channel. The counterflow velocity is varied over the course of a separation so that analytes with different electrophoretic mobilities enter the separation channel at different times and are detected as moving boundary, stepwise increases in the detector response. The resolution of a GEMBE separation is thus dependent on the rate at which the counterflow velocity is varied (rather than the length of the separation channel), and relatively high resolution separations can be performed with short microfluidic channels or capillaries. In this paper we describe an implementation of the GEMBE technique in which a very short (2.5-3.5 mm) capillary or microchannel is used as both the separation channel and a conductivity detection cell. Because the channel is so short, only a single moving boundary "step" is present in the channel at any given time, and the measured current through the channel can therefore be used to give a signal comparable to what is normally generated by more complicated detector arrangements. A theoretical description of the new technique is given along with simulation and experimental data relevant to the optimization of the method parameters such as channel length, counterflow acceleration, and applied field strength. A key theoretical prediction is that although this technique is expected to be a factor of 10 or 20 slower than conventional capillary zone electrophoresis, separation times of the order 1 s or less can still be achieved, making it applicable for ultrahigh-throughput analyses when implemented in a multiplexed format.
梯度洗脱移动边界电泳（GEMBE）是一种最近描述的用于在短（1 - 3厘米）毛细管或微通道中进行电泳分离的技术。使用GEMBE时，分析物的电泳迁移与分离缓冲液通过分离通道的整体逆流相反。在分离过程中，逆流速度会发生变化，以便具有不同电泳迁移率的分析物在不同时间进入分离通道，并被检测为移动边界，即检测器响应的逐步增加。因此，GEMBE分离的分辨率取决于逆流速度变化的速率（而不是分离通道的长度），并且可以使用短的微流体通道或毛细管进行相对高分辨率的分离。在本文中，我们描述了GEMBE技术的一种实现方式，其中使用非常短（2.5 - 3.5毫米）的毛细管或微通道作为分离通道和电导检测池。由于通道非常短，在任何给定时间通道中仅存在一个移动边界“台阶”，因此通过通道测量的电流可用于给出与更复杂的检测器配置通常产生的信号相当的信号。给出了新技术的理论描述以及与方法参数优化相关的模拟和实验数据，如通道长度、逆流加速度和施加场强。一个关键的理论预测是，尽管预计该技术比传统毛细管区带电泳慢10倍或20倍，但仍可实现1秒或更短的分离时间，当以多路复用形式实施时，使其适用于超高通量分析。
