Biomedical Engineering – From Theory to Applications

Column Coupling Electrophoresis in Biomedical Analysis
the analytes they make tools that can be effectively on-line combined on the column
coupling chip in two general ways (Kaniansky et al., 2000; Wainright et al., 2002; Bodor et
al., 2002) (i) ITP, concentrating the sample constituents into a narrow pulse is intended,
mainly, as a sample injection technique for ZE; (ii) ITP, while concentrating the analyte and
some of the matrix constituents into a narrow pulse, serves mainly as a sample clean-up
technique and removes a major part of the sample matrix from the separation system before
the final ZE separation. For the separation mechanism of ITP-ZE in microchip format see
Fig. 2, that is principally the same for the capillary and microchip format. MCE provided
with the column-coupling (CC) configuration of the separation channels for the ITP-ZE
separations is illustrated in Fig. 11. Different volumes of the sample channels (S1, S2) serve
for a low or large volume injection depending on analyte and matrix concentration. At this
scheme, the contact conductivity detector is used, nevertheless, other common detectors
such as UV-VIS absorbance photometric detector, and especially LIF detector can be
successfully applied, see e.g. (Belder, 2006).
Fig. 11. MCE provided with the column-coupling (CC) configuration of the separation
channels. CC poly(methylmethacrylate) chip provided with the conductivity detection cells.
Details: C3 = terminating electrolyte channel; S1 and S2 = 9000 and 950 nL sample injection
channels, respectively; W = an outlet hole from the chip channels to a waste container; C1 =
first separation channel (3050 nL volume; 76x0.2x0.2 mm (length, width, depth)) with a
platinum conductivity sensor (D1); C2 = second separation channel (1680 nL volume;
42x0.2x0.2 mm) with a platinum conductivity sensor (D2). Reprinted from ref. (Kaniansky et
al., 2003), with permission.
3.2.1.2 ITP-GE
ITP-GE is proposed for the special category of separations where high molecular
compounds are separated from each other in presence or absence of matrix constituents
(Huang et al., 2005). A microchip for integrated ITP preconcentration with GE separation
enables to decrease the detectable concentration of biopolymers such as sodium dodecyl
sulfate (SDS)-proteins. Each channel of the chip is advantageously designed with a long
sample injection channel to increase the sample loading and allow stacking the sample into
a narrow zone using discontinuous ITP buffers. The preconcentrated sample is separated in
GE mode in sieving polymer solutions. All the analysis steps including injection,
preconcentration, and separation of the ITP-GE process are performed continuously,
controlled by a high-voltage power source with sequential voltage switching between the
analysis steps. Without deteriorating the peak resolution, the integrated ITP-GE system can
result in a decreased detectable concentration of tens-fold compared to the GE mode only.
The picture of the ITP-GE microchip and the protocol of the ITP-GE procedure on the
microfluidic device are illustrated in Fig. 12.
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