View - ResearchGate

310 Heegaard et al.
characteristics may be desired, e.g. when studying metal-ion-binding proteins
(40,41). Calcium ions will, for example, precipitate in phosphate buffers. Very
reliable results may instead be obtained with HEPES buffers that have minimal
cation-binding (42). In work involving, for example Ca 2+ , it may be necessary
to use chelating agents such as ethylenediaminetetraacetic acid (EDTA) in the
washing solutions to remove all divalent cations between runs. The magnitude
of the EEO flow will be a sensitive indicator of the amount of immobilized
cations in such experiments.
The interplay between sample solution and electrophoresis buffer also
requires attention. Conductivity differences may be detrimental but may also
be exploited to increase detection limits by taking advantage of stacking
phenomena. It is important to realize that even though considerable increases
in detection limits may be achieved by dissolving the analyte in a sample
buffer with lower (typically 1/10 diluted electrophoresis buffer) conductivity
than the electrophoresis buffer (43,44), the resulting temperature increase in the
sample zone may be very high leading to, for example, heat-induced partial or
complete denaturation or the induction of other artifacts, such as, aggregation
of the protein analyte (22) (see Fig. 2). Conversely, when the conductivity of
the sample is higher (e.g. because of a high salt content), analyte peak broadening
is to be expected. Also, in any CE experiment where buffer and sample
conductivity is not the same, the precise concentration of the analyte in the
sample zone is bound to be different from the concentration in the sample and
will change during the initial electrophoresis steps. This complicates affinity
experiments where the exact concentration of analyte during the run is required
for the subsequent calculations.
In addition to conductivity differences, the correspondence of pH in the
sample solution and in the electrophoresis buffer also warrants attention because
the crossing of the pH boundary created upon initiation of electrophoresis may
lead to analyte aggregation and precipitation.
Finally, the vial strategy should be considered for two main reasons: one is
that repeated electrophoresis from the same buffer vial will lead to so-called
buffer depletion, (a change, caused by electrolysis, in the ionic composition of
the anodic and cathodic buffer solutions) leading to changes in mobility when
the electrolyzed buffer is used as a running buffer. Thus, fresh buffer should
always be used to replenish the electrophoresis buffer, for example, by using
different reservoirs for running and for rinsing. This will ensure a reproducible
composition of the buffer inside the capillary. Another detail regarding vial and
washing strategies is that in affinity electrophoresis with ligand addition to the
electrophoresis buffer, it is normally not the intention to introduce ligand into
the sample solution. Carry over of ligand into the sample solution when sample
injection follows immediately after washing the capillary with the ligand-