Biomedical Engineering – From Theory to Applications

Column Coupling Electrophoresis in Biomedical Analysis
detection of the ITP zones, conductivity detection technique has a limited applicability in
the CZE separations (often measurements of small conductivity changes due to the zones
on a relatively high conductivity background of the carrier electrolyte) (Ölvecká et al.,
2001; Kaniansky et al., 2000).
3.1.1.2 ITP-ITP
The ITP-ITP combination represents the simplest possibility how to combine CE
techniques. For the general instrumental scheme valid also for ITP-ITP, see Fig. 1. In the
ITP–ITP mode both preseparation (wider) and analytical (narrower) capillaries are filled
with (i) the same leading electrolyte (one-dimensional ITP) or (ii) different electrolytes
(two-dimensional ITP) (Flottmann et al., 2006; Bexheti et al., 2006; Mikuš et al., 2006b;
Kubačák et al., 2006a, 2006b, 2007). The ITP separation in a concentration cascade,
introduced into conventional CE by Boček et al., (Boček et al., 1978) enhances the
detectabilities of the separated constituents from the response of the conductivity
detection due to well-known links between the concentration of the leading electrolyte
and the lengths (volumes) of the zones (Marák et al., 1990)
The first ITP stage of the ITP-ITP combination can apply all benefits as they are described
for the ITP stage of the ITP-CZE combination in section 3.1.1.1. On the other hand, the ITP-
ITP technique can take the highest advantage of the hyphenation with the MS detection
(Tomáš et al., 2010). It is because of an intrinsic feature of ITP to produce pure analyte zones,
i.e. those in which the analyte is accompanied only with counter ion, in the isotachophoretic
steady state. In this way, the maximum response of the MS detector can be obtained for the
analyte. Therefore, the ITP-ITP-MS hyphenation seems to be one of the most promissing
methods for the fully automatized biomedical analyses such as pharmacokinetic studies,
metabolomics, etc. An economic aspect of the ITP-ITP-MS method in comparison with the
HPLC-MS method for the ionic compounds is apparent.
3.1.1.3 ITP-CEC
Another approach in the column coupled electrophoresis is the use of ITP sample focusing
to improve the detection limits for the analysis of charged compounds in capillary
electrochromatography (CEC). Besides this, the on-line isotachophoretic stage can serve also
for a loadability enhancement (due to a large inner diameter of the ITP capillary). Both of
these effects are then responsible for a dramatic reduction of the sample concentration
detection limits through simultaneous acting of (i) large volume injection and (ii) analyte
stacking (Mazereeuw et al., 2000).
In the ITP-CEC combination (Fig. 4), the open ITP mode must be applied because of the
demands of the second stage (CEC) that is based on the EOF action. A coupled-column
set-up can be used, in which counterflow ITP focusing is performed, and the separation
capillaries are connected via a T-junction. For the schematic representation of the ITP–
CEC procedure see Fig. 5. From the application point of view, the first ITP stage is
advantageous especially for the injection of large volumes (tens of microliters) of diluted
samples. When a very large sample is introduced, however, the focusing time of the
sample often exceeds the migration time to the outlet of the ITP capillary. By applying a
hydrodynamic counterflow (applicable in the hydrodynamically open CE systems) the
ITP focusing will continue while extending the migration towards the outlet of the ITP
capillary. Although the hydrodynamically open CE systems have the advantage of
application of the supporting flows (counterflow, electroosmotic), it must be realized that
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