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

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volume. As a rule of thumb, the sample plug length should
be less than 1 to 2 % of the total length of the capillary. This
corresponds to an injection length of a few millimeters
(or 1 to 50 nl), depending on the length and inner diameter.
This is an advantage when sample volumes are limited since
5 µl of sample is sufficient to perform numerous injections.
Conversely, the small volumes seriously increase sensitivity
difficulties for dilute samples.
Sample overloading can have two significant effects, both
detrimental to resolution (figure 50). First, injection lengths
longer than the diffusion controlled zone width will proportionally
broaden peak width. Secondly, it can exacerbate
field inhomogeneities and distorted peak shapes caused by
mismatched conductivity between the running buffer and
the sample zone.
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Figure 50
Effect of sample overloading on peak
shape 27
Starting zone widths: a = 0.6 cm,
b = 2.0 cm,
c = 3.0 cm
Quantitative sample injection can be accomplished by a
number of methods. The two most common are hydrodynamic
and electrokinetic (figure 51). In either case, the
sample volume loaded is generally not a known quantity,
although it can be calculated. Instead of volume, the
quantifiable parameters are pressure/time for hydrodynamic
injection, or voltage/time for electroldnetic injection, as
described in detail in the next two sub-sections.
4.1.1 Hydrodynamic injection
Hydrodynamic sample injection is the most widely used
method. It can be accomplished by application of pressure
at the injection end of the capillary, vacuum at the exit end
of the capillary, or by siphoning action obtained by elevating
the injection reservoir relative to the exit reservoir (figure
51a,b,c). With hydrodynamic injection, the quantity of
sample loaded is nearly independent of the sample matrix.
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