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312 Heegaard et al.
sample. A final note regarding the buffer vials is that a hydrodynamic force
(siphoning) will be added to electrophoresis and EEO during a separation if
the capillary ends are at different fluid heights, and this may be detrimental to
efficiencies (21). Thus, it is important to ensure that inlet and outlet vial buffer
levels are equal.
3.3. Running Conditions
After appropriate conditioning/washing, and pre-rinsing, the affinity electrophoresis
experiment is initiated by injecting the sample and applying a current.
The controllable parameters here include sample injection mode and settings for
time/current/field strength, and in some cases sample temperature. In addition,
there are choices to be made regarding constant current/voltage/power, rise
times, detection mode, run time and capillary temperature control.
With regard to the sample solution temperature, this is controllable in some
instruments by an external circulating water bath, and this may be very helpful in
instances when studying protein folding–unfolding processes (45) and when the
sample stability or pre-incubated binding interaction is temperature dependent.
For sensitive experiments, it is advisable to control the actual temperature with
a temperature probe into a sample vial. When working with different sample
temperatures, it is also worth considering that solution viscosity, and thus
injected volume in pressure injection modes, is changing with temperature. The
viscosity of aqueous solutions increases with decreasing temperature. The peak
area of a marker (e.g. a non-interacting peptide) may be used to normalize such
injection volume fluctuations. Because sample volumes usually are in the 5- to
50 L range (with injected volumes in zone electrophoresis usually being in the
1- to 15 nL range), another issue that merits attention is sample evaporation.
Again, an internal calibrant may be used to correct for changes in analyte
concentration caused by evaporation, but for larger time series where maybe
many hundred injections are going to be performed from the same solution, the
use of a protective layer of light mineral oil on top of the sample (as known
from PCR experiments) will prevent evaporation (46).
In zone electrophoretic applications, the sample volume injected is normally
not much more than 1–5% of the total capillary volume which usually is
1–2 L. Injection may be performed by positive or negative pressure (hydrodynamic
injection) or by current. The latter mode has the disadvantages of being
selective (relatively more of high mobility components will be sampled), of
altering the electrolyte composition in the sample and of being less reproducible
than hydrodynamic injections (21). There are few reasons to use this sampling
method in free solution electrophoresis except maybe to enrich for a specific
high mobility analyte component.