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

Principles
k' H N
H, µm
0.001 0.58 1.7 × 10 6
0.005 1.00 1.0 × 10 5
0.010 1.54 6.5 × 10 5
0.050 6.78 1.5 × 10 5
0.100 15.7 6.4 × 10 4
Table 7
Impact of adsorption on efficiency 1
D = 5 × 10 -5 cm 2 /s v EOF
= 1 mm/s
l = 50 cm L = 60 cm
20
18
16
14
12
10
8
6
4
2
kd = 10 s -1
Dm = 10 -4 cm 2 /s
k' = .07
k' = .03
k' = .01
k' = .002
k' = .001
.010 .020 .030 .040 .050 .060 .070 .080 .090 .100
V, cm/s
Figure 16
Effect of protein-wall interactions on HETP 1
practice, protein separations typically exhibit capacity
factors between 0.01 to 0.1. Thus, plate counts between 1 to
5 × 10 5 may be expected.
There are a variety of strategies employed to reduce solutewall
interactions. Among these, a few are very simple to
implement and often yield exceptional results. Increasing
the concentration of the buffer, for example, decreases
solute interactions by reducing the effective surface charge.
High ionic strength also decreases the EOF, thereby increasing
the residence time of solutes in the capillary. The benefit
of these effects is illustrated in the peptide digest separation
of figure 17. Again, this approach is somewhat limited by
the increased current and subsequent Joule heating. Use
of zwitterionic buffer systems are an alternative.
Another approach to limit adsorption is to operate at the
extremes of pH. At low pH (< 2 to 3) the silanol groups of
fused silica will be essentially protonated and uncharged.
While EOF will be nearly zero, since proteins (and other
species) will also be protonated and positively charged, they
will migrate toward the cathode. Conversely, at high pH (> 9
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