Biomedical Engineering – From Theory to Applications

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Biomedical Engineering – From Theory to Applications
Fig. 3. Graphical illustration of the principle of the electronic cutting of the zone of interest
in the ITP stage of the ITP-CZE combination. L = leading ion, T = terminating ion, X =
matrix compound(s), Y, Z = analytes, R = resistance. Reprinted from ref. (Ölvecká et al.,
2001), with permission.
The principle of this hyphenated technique consists from well-defined preconcentration
(concentration LODs could be reduced by a factor of 10 3 when compared to conventional
single column CZE) and preseparation (up to 99% or even more interfering compounds
can be isolated (Danková et al., 1999)) of trace analytes in the first, wider, capillary
(isotachophoretic step) and subsequently a cut of important analytes accompanied with a
segment of the matrix, leading or terminator enters the second, narrower, capillary for the
final separation by CZE (Fig. 2, Fig.3). The presence of this segment results from the fact
that we do not want to lose a part of the analyzed zones and we must make a cut
generously. The zone of this segment survives for a certain time during the CZE stage and
this mean that ITP migration continues also in the second capillary for some time and it
influences strongly the results of the analysis, especially the detection times of analytes
used for identification of the analytes in CZE separations (Busnel et al., 2006; Gebauer et
al., 2007; Mikuš et al., 2006a). From this is clear that it is important in an ITP-CZE
combination to choose suitable electrolyte systems and find the optimum time to switch
the current from the preseparation capillary to the separation capillary (Křivánková et al.,
1995).
The ITP-CZE technique appeared to be very useful especially for the common universal
detectors producing relatively low concentration LODs (UV-VIS photometric detector). It
is because such method provides probably one of the most acceptable ratio simplicitycost:
universality-concentration LOD in comparison to other column coupling methods
and detection systems. This suggestion is supported by many advanced applications of
the ITP-CZE-UV method in the pharmaceutical and biomedical field (Marák et al., 2007;
Mikuš et al., 2008a, 2008b, 2008c, 2009). Jumps in voltage (conductivity) between
neighboring zones result in permanently sharp boundaries between zones (Fig. 3) that is
extremely convenient for the conductivity detection in ITP. Although convenient to the