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

Modes
Crosslinked polyacrylamide, a widely used matrix, is usually
polymerized in situ and not removed from the capil- lary.
Preparation of these gels requires extreme care. Too rapid
polymerization, use of non-degassed solutions, or impure
chemicals often lead to bubble formation or unstable gels.
A potential disadvantage of crosslinked polyacrylamide is
its rigid nature. Dirty samples, clogging of the ends, or
bubble formation during use may make the capillary unusable.
Care must be exercised to prevent these from occurring.
With proper use, numerous injections on a single
capillary are possible. In this regard, the rigidity of the gel
precludes the use of hydrodynamic sample injection.
0.001 AU
40 50 60
Time [min]
64 65
Figure 42
CGE separation of polydeoxythyamidylic
acid mixture, p(dT) 20-160
using crosslinked
polyacrylamide
21
Conditions: Bis-crosslinked polyacrylamide
(6 % T, 5 % C), 100 mM Tris,
25 mM borate, 7 mM urea, pH 7.6,
E = 200 V/cm, i = 8.2 mA, l = 100 cm,
id = 75 mm, l = 260 nm, polyacrylamide
coated capillary
*
Linear polymers offer an alternative to the crosslinked gels.
Since they are essentially polymer solutions, they are much
more flexible. The linear polymer solutions may also be
polymerized in situ, but it is not necessary. Pre-polymerized
polymer can be dissolved in buffer and hydrodynamically
loaded into the capillary. For polyacrylamide, a wide range
of gel concentrations can be used (that is, below 1 % to
more than 20 %). Generally, the polymer concentration
necessary is inversely proportional to the size of the analyte.
With low viscosity polymer solutions, pressure can be used
for sample injection and they can be repeatedly filled into
the capillary (depending on concentration and viscosity).
They are also less susceptible to bubble formation and other
failures. While they possess more stability in these respects,
the less viscous the gel the more dependent the system is on
the integrity of the wall coating (if used). With either type,
the capillary wall is usually coated to eliminate EOF.
Resolution and efficiency in CGE are identical to that in CZE
since they are both “zonal” electrophoretic techniques. One
notable difference is the ultra high efficiency achievable for
DNA separations. Figure 42 illustrates that over 10 7 plates/m
(*) can be realized for single-stranded oligonucleotides
using crosslinked polyacrylamide. As in CZE, selectivity in
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