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

Modes
Absorbance 214 nm
0.05
0.04
0.03
0.02
0.01
0
-0.01
7 8 9 10 11 12
Time [min]
50 ˚C
40 ˚C
30 ˚C
20 ˚C
Figure 24
Temperature-induced structural changes
of horse heart myoglobin 10
Conditions: 0.1 M Tris-25 mM boric acid,
pH 8.6, constant current = 9.8 mA,
l = 50 cm, L = 57 cm, id = 75 mm,
l = 214 nm
In addition, as the temperature is increased, resolution is
reduced, presumably due to decreased equilibrium constant
and limited solute-CD interaction.
3.1.1.5 Temperature
Although the primary purposes of thermostatically controlling
capillary temperature are to maintain constant temperature
and to remove Joule heat, temperature control can
also be used as a parameter in optimizing a CZE separation.
Elevated or reduced temperatures alter viscosity, EOF, and
analysis time. As shown in the cyclodextrin example (figure
23d), it can also be used to affect chemical equilibria and
kinetics. Temperature can also be used to affect protein
conformation or protein-DNA interactions, for example.
To this end, the temperature-induced structural changes of
myoglobin are shown in figure 24. This behavior has been
attributed to the possible reduction of the iron coordinated
to the heme group.
3.1.2 Capillary wall modifications
CZE is an important separation technique for both small and
macromolecular solutes. From basic theory it is expected
that macromolecules such as proteins would yield very high
efficiencies (N >10 6 ) due to their low diffusion coefficients.
It has been found, however, especially for proteins, that
interaction with the capillary surface greatly reduces
efficiency. These interactions can be ionic and/or hydrophobic
in nature. Such difficulties are not surprising considering
the variability of proteins with regard to charge, hydrophobicity,
size, and dynamic nature.
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