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

Modes
1
2
3 45 6
16 20 24 28 32 36
27 31 35 39 43 47
Time [min]
Figure 25
Aryl pentafluro-coated capillaries to
improve protein separations. Coated
capillary above, bare fused silica below 11
Peaks: 1 = lysozyme, 2 = DMSO (EOF marker),
3 = bovine pan- creatic trypsinogen, 5 = whale
myoglobin, 6 = horse myoglobin, 7 = human
carbonic anhydrase, 8 = bovine carbonic
anhydrase B.
Conditions: 200 mM phosphate, 100 mM KCl,
pH 7, E = 250 V/cm, id = 20 mm,
l = 219 nm
7
8
a)
b)
As described in section 2.3, studies have shown that
protein-wall interactions with k' values that would be
considered of no significance in LC can have an appreciable
effect in CE. Such interactions often result in peak tailing or
even total retention in the capillary.
Without explicit wall modification, use of pH extremes is
very effective in reducing ionic interactions. A possible
limitation of this approach is the alteration of protein
structure at non-biological pH values. High ionic strength
buffers can limit ionic interactions, although ultimately
limited by Joule heating. While narrow-bore capillaries can
be beneficial with respect to heating, protein-wall interactions
are exacerbated by the high surface area-to-volume
ratio capillary.
Capillary wall modification is an alternative to limit solute
adsorption. Two fundamental approaches have been taken:
a) permanent modification by covalently bonded or physically
adhered phases; and b) dynamic deactivation using
running buffer additives. Both approaches have been somewhat
successful, although no single method is clearly
superior.
3.1.2.1 Bonded or adhered phases
A number of permanent wall modifications are described in
table 12. Notably, silylation followed by deactivation with
a suitable functional group has been the most widely used
approach. Deactivation can be accomplished with such
varied species as polyacrylamide, aryl pentafluoryl groups,
or polysaccharides. The electropherograms in figure 25
show the type of improvement that can be expected for the
separation of proteins using coated capillaries. Unfortunately,
the siloxane bond (Si-O-Si) is stable only between
pH @ 4 and 7 and hydrolysis usually limits long term
stability.
55