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

Instrumentation/Operation
mAU
140
120
100
80
60
40
20
140
120
100
80
60
40
20
0
Figure 67
Improved migration time reproducibility
using buffer replenishment
Overlay of runs 5, 10, and 15
a) replenish after every 5 runs
b) no replenishment
a)
3.0 3.5 4.0 4.5 5.0 5.5
b)
3.0 3.5 4.0 4.5 5.0 5.5
Time [min]
system, the volume of the reservoirs, and whether the
capillary conditioning washes are flushed into the exit
reservoir (not recommended). For these reasons, frequent
replacement of buffer is recommended. An example of the
benefit of frequent buffer replenishment is shown in figure
67.
The replenishment system operates by emptying the contents
of the reservoirs into a waste bottle and then refilling
them with fresh buffer. With a large buffer replenishment
system minimal vial locations in the autosampler are needed
for buffer, leaving more locations for samples. In addition,
a large reservoir affords the capability to run automated
analysis for a long period of time (over the weekend, for
example).
4.4.4 Buffer leveling
As described in chapter 2, maintaining equal liquid levels is
important to both efficiency and migration time reproducibility.
Siphoning from one reservoir to the other will superimpose
laminar flow into the system. This is not only detrimental
to reproducibility but also to the efficiency. The
influence of non-level reservoirs is dependent on the diameter
and length of the capillary and the viscosity of the
buffer. Clearly, wide-bore, short capillaries run at elevated
temperatures would exhibit the most deleterious effects.
It has been shown that a height difference of 2 mm in a
50-µm id capillary can change migration time 2 to 3 %. This
increases to about 10 % in a 100-µm id capillary.
An automated buffer leveling system would accurately
deliver liquid to a user-specified level during replenishment.
It can also be used to re-level liquids in the running reservoirs
caused by EOF, without emptying them first.
112