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Affinity Capillary Electrophoresis 309
The starting point in electrophoresis buffer selection is the condition that
best mimics the environment in which it is interesting to characterize the interaction
in question. Thus, for serum proteins, an isotonic buffer (corresponding
to 154 mM NaCl), pH 7.4 will be appropriate, while for proteins functioning in
specialized sites, e.g. in kidney compartments, at infectious sites or intracellularly,
very different conditions may be appropriate. If this first choice of buffer
turns out to be incompatible with analysis one may try to modify it, (e.g. if
protein adsorption is the problem, by modifying pH in small steps to determine
the smallest pH-shift from the ideal value that allows for a reproducible analysis
with full recovery of the analyte (31)) or by adding various non-ionic detergents
to disaggregate interacting hydrophobic patches (32). High ionic strength may
by itself be sufficient to counteract wall interactions and increase resolution
(33). Also, ion-pairing agents (34,35), as known from reversed phase high
pressure liquid chromatography (RP-HPLC), may be used to the same effect as
long as it is ensured that these agents do not themselves interact with analytes
or ligand additives, and that the current increases that are bound to occur with
increased charged ions in the buffer, are not detrimental for the temperature
inside the capillary. This may be an issue for easily denatured proteins. Some
strategies to counteract wall interactions rely on utilizing the pH hysteresis
effect of fused silica (36). This is conveniently achieved by an acid pre-rinse
solution (for example, 0.1 M HCl instead of 0.1 M NaOH), which will diminish
capillary wall deprotonation and negative charge at the ensuing neutral pH
analysis (37,38).
The single most important analyte parameter influencing electrophoretic
mobility is charge, i.e. electrophoresis buffer pH (5,6). The buffer choice is
also specifically influenced in binding experiments with ligand addition to the
electrophoresis buffer by solubility and other ligand characteristics in particular
buffers. When deciding on the pH of a separation, all the usual buffer considerations
such as buffer capacity and buffering range apply. In addition, some
CE-specific features such as the UV-transparency and heat capacity influence
the choice of electrophoresis buffer. Also, it is important to remember that buffer
components such as ions added may adhere in a charge-dependent fashion to
the inner capillary surface. It is always instructive to watch the EEO flow for
changes as an indicator of immobilized wall-charge changes. In special cases,
for instance, when performing low-temperature electrophoresis, the viscosity
characteristics of the electrophoresis buffer also become important (39).
The UV-transparency of buffers is extremely important for low-wavelength
(200 nm) detection, which is most often employed in work with proteins.
Even under the best of conditions, the polypeptide limit-of-detection (LOD)
rarely exceeds 1 M. A high UV-absorbance by the buffer decreases the
linear dynamic range of the detector and thus peak heights. Specific buffer