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Affinity Capillary Electrophoresis 307
sure that temperature conditions are constant in all the experiments in a series.
Owing to the decrease in viscosity with temperature and the increase in current,
the observed peak migration is extremely dependent on the temperature conditions.
Therefore, the uniformity and consistency of the cooling are of great
importance for ACE experiments. Also, the UV-absorbance of most buffers
is dependent on the temperature of the buffer. The capillary length is chosen
to provide enough separation with minimal diffusion (peak broadening) and
enough resistance to minimize current. It should also be considered that there
is a relation between the stability of a molecular complex and the separation
time. Short-lived, low-affinity pre-incubated complexes require short separation
times (capillaries with short effective lengths i.e., distance to the detector point
and/or high field strengths) to be detected before they dissociate. As a rule,
to ensure 10% or less-complex dissociation during separation, the dissociation
rate constant of the complex should be less than 0.105/t, where t is the time
it takes to separate the peaks (24). Sometimes, it will therefore be advantageous
to inject and separate pre-equilibrated samples from the short end of
the capillary or use custom-made apparatus on microchips and/or flow-gated
capillaries to achieve separations as short as 1sorlower. This also enables
on-line immunochemical monitoring of biofluids (25–28). There are, however,
practical limits to how short a capillary can be fitted into commercial instruments,
and decreasing resolution also determines how short a capillary can be.
In many cases, when studying low-affinity interactions, the mobility shift ACE
approaches (c.f. Subheading 6) may instead be considered.
Very narrow capillaries will allow very high field strengths to be applied and
will thus increase separation efficiency – however, at the expense of detection
limits. Regarding the handling of capillaries, it is sensible to pay attention to the
cut edges of the capillary ends; the less frayed and irregular these can be made,
the less is the risk of carry-over, irreproducible pressure injection volumes and
capillary blockage (see Fig. 1).
Having taken into account stable temperature and current and sufficient
detection path length, the by far most prevalent problem is the recovery of
protein analytes in uncoated fused silica capillaries at the neutral pH conditions
that normally will be favoured for binding experiments.
Coated capillaries may overcome some protein adsorption problems and
come in many different versions, but overall such capillaries have not been
used much, probably because any kind of coating whether being dynamic or
static (29) will be associated with its own set of problems. Also, the great
feature of electroendosmosis (EEO)-assisted electroseparations is that it makes
all analytes analyzable in one operation without changing polarity although
various coatings may eliminate or reverse the EEO flow. Coated capillaries
also generally have shorter life-spans than plain capillaries.