High performance capillary electrophoresis - T.E.A.M.

Modes
and resolution with respect to pI difference is given by:
dpH
( dx )
( )
1/2
DpI = 3 (27)
E
-dm
dpH
where D = diffusion coefficient (cm 2 /s)
E = electric field (V/cm)
dpH/dx = pH gradient at the zone
dm/dpH = mobility slope at the pI (which
depends on the charge of the
protein near its pI).
As described by equation (27) high resolution is obtained
with high electric fields, a high mobility slope at the isoelectric
point, and a shallow rate of change of pH.
EOF needs to be reduced or eliminated in CIEF since the
flow could flush the ampholytes from the capillary before
focusing is complete. Reduction of EOF can be accomplished
by the use of dynamic or covalent coatings. The
dynamic coatings have the advantage of simplicity, but
obtaining reproducible EOF is often difficult. The coating,
either dynamic or covalent, is also helpful in limiting protein
adsorption to the capillary walls.
Since the solute is loaded on to the capillary during filling
with the amphoteric solution, it is possible to load significantly
larger volumes than in most other CE modes. Precipitation,
which results from very high protein concentrations
within the zones, is usually the limiting factor in sample
loading.
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