Biomedical Engineering – From Theory to Applications

Column Coupling Electrophoresis in Biomedical Analysis
sample load capacity (30 L sample injection volume is typical) due to the large internal
diameter of the preseparation capillary (800 m I.D. is typical) (Kaniansky & Marák, 1990;
Kaniansky et al., 1993). The commercially available CE-CE systems have a modular
composition that provides a high flexibility in arranging particular moduls in the separation
unit. In this way, it is possible to create desirable CE-CE combinations such as (i) ITP-ITP, (ii)
ITP-CZE, (iii) CZE-CZE, etc., capable to solve wide scale of the advanced analytical problems
(see Fig. 1). Although such combinations require the sophisticated instrument and deep
knowledge in the field of electrophoresis, the coupled CE methods are surely the most
effective way how to take/multiply benefits of both CE techniques coupled in the columncoupling
configuration of separation unit. The basic instrumental scheme of the column
coupled CE-CE system shown in Fig. 1 is properly matching with hydrodynamically closed
CE modes where effective electrophoretic mobility is the only driving force of the separated
compounds. On the other hand, when additional supporting effects such as counterflow,
electroosmotic flow etc. must be employed, appropriate modifications of the scheme in Fig. 1
are made. Such modified instrumental schemes are attached into the sections dealing with IEF
or CEC coupled techniques (3.1.1.3 and 3.1.1.5) that are principally applicable only in
hydrodynamically open CE mode (Mikuš et al., 2006; Danková et al., 2001; Busnel et al., 2006).
Fig. 1. CE-CE method in column coupling configuration of the separation units for the direct
analysis of unpretreated complex matrices sample, basic instrumental scheme. On-line
sample preparation: removing matrices X (ITP, CZE), preseparation (ITP, CZE) and/or
preconcentration (ITP, stacking) and/or derivatization (with stacking) of analytes Y, Z in the
first CE stage (column C1). Final separation: baseline separation of Y and Z in the second CE
(ITP, CZE) stage (column C2). Reprinted from ref. (Tekeľ & Mikuš, 2005), with permission.
C1 – preseparation column, C2 – analytical column, B – bifurcation block for coupling C1
and C2, D – positions of detectors.
3.1.1.1 ITP-CZE
Although all electrophoretic methods can be mutually on-line combined, the biggest
attention was paid to the ITP-CZE coupling, introduced more than 20 years ago by
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