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Affinity Capillary Electrophoresis 313
If temperature in a sample-stacking zone is a concern, one may program a
step-wise increase to ensure electrophoretic transport of the analyte into the
electrophoresis buffer before the full field strength is applied (see Fig. 2). The
choice of electrical parameters is otherwise an interplay between efficiency,
time and induced temperature characteristics of the electrophoresis buffer
(and thus on the efficiency of the cooling system). If as high a field
strength as possible is desirable, one may use an Ohm’s law plot to estimate
the breakthrough-current (where the linearity of current as a function of
applied potential is lost because the resistance drops with uncontrollable
increase in temperature caused by inadequate Joule heat dissipation) (21,47).
Performing separations under constant current settings has the advantage that
the amount of induced Joule heat is constant. With constant field strength,
more constant migration times will be obtained. However, there will be current
and thus temperature fluctuations. These are usually of minor importance if
the temperature is kept constant and the conductivity in sample solution and
electrophoresis buffer is not too different.
Detector choices depend on the nature of the compounds involved in the
affinity interaction and the scope of the analysis. In UV-absorbance detection,
the concentration LOD is only in the low micromolar range for polypeptides
(see Note 2). This confers a problem when measuring binding of lowconcentration
analytes and molecules involved in strong binding interactions.
In these situations, much lower detector sensitivity is required. Labelling of
the interacting molecules with fluorescent probes will increase the sensitivity
of the system, sometimes down to sub attoM concentration LOD (48), but
also modifies the structure of the analyte covalently possibly changing analyte
electrophoretic mobility and binding behaviour. Laser-induced fluorescence
detection principles are reviewed in (49). The types of fluorescent probes
available are diverse, and thus in many cases, it is possible to avoid the interfering
effect caused by the labelling. One example is to carbohydrate-tag an
analyte with fluorescein-thiosemicarbazide as example in studies of the binding
interactions of rHLA–DR4 complex with influenza virus hemagglutinin peptide
ligand. The fluorescein-thiosemicarbazide probe is attached at the carbohydrate
moiety of the protein complex which is not involved in the interaction (32).
Alternatives to the commonly used UV-absorbance and laser induced fluorescence
(LIF) detectors are electrochemical detectors which have proven advantageous
when analyzing for metal ions and small inorganic molecules in
biological fluids (50), but which are difficult to use in conjunction with physiological
buffers. Radioactivity based detectors may be very sensitive (51) but
entail the use of non-standard detector equipment and require labelling of
analytes.