Page 73 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

P1: LDK Revised Pages
Encyclopedia of Physical Science and Technology EN002E-79 May 17, 2001 20:28

Capillary Zone Electrophoresis 359

length should not exceed 5% of the total capillary length. The small internal diameters of fused silica capillar-
Sample zone sharpening can be achieved by preparing ies have been the key to high-resolution CE separations.
the sample in an electrolyte of lower conductivity than The smaller the capillary bore, the greater the surface-to-
the analysis buffer. Under these conditions, there is a dis- volume ratio and the more efficiently heat is removed from
continuity in field strength at the sample:buffer boundary the tube. The optimal capillary inside diameter (i.d.) for
such that ions migrating rapidly from a region of higher most applications is 50 to 75 µm. Capillaries with smaller
field strength become focused at the boundary. This fo- diameters are subject to plugging, and the high surface-
cusing or stacking effect not only produces narrow zones to-volume ratio increases the risk of adsorption. Capillar-
for increased resolution, but also increases zone concen- ies with larger internal diameters may exhibit significant
tration for enhanced sensitivity. Sensitivity enhancement loss in resolution due to thermal effects, and may require
is directly proportional to the ratio of sample to buffer operation at low field strengths or with low-conductivity
conductivity. Sample zone sharpening can also be accom- buffers.
plished by transient isotachophoretic preconcentration, as
discussed below.
D. Adsorption (h ads )

B. Diffusion and Electrodispersion (h diff+cond ) Wall interactions have been the greatest obstacle to achiev-
ing satisfactory resolution and reproducibility in capillary
Band broadening due to axial diffusion should be reduced electrophoresis, particularly in zone electrophoresis and
by shortening analysis time (i.e., by operating at high field isoelectric focusing. Approaches to minimizing or elimi-
strengths with short capillaries). On the other hand, higher nating adsorption include use of buffer additives and dy-
field strengths will generate more Joule heat, which in- namic or covalently-coupled capillary coatings.
creases diffusion rates. Electrodispersive band broaden-
ing arises from conductivity differences between the zone
and the background electrolyte. If this difference is large,
diffusion at one boundary of the zone is neglible due to III. CAPILLARY ELECTROPHORESIS
the zone-sharpening effect caused by the discontinuity in INSTRUMENTATION
field strength. In this case, the peak will be asymmetric;
that is, it will exhibit fronting or tailing depending on A variety of commercial CE instruments are available,
whether conductivity of the zone is greater or less than from simple modular systems consisting of a power sup-
that of the background electrolyte. This phenomenon is ply, detector, and injection device to fully integrated au-
readily observable when analyzing highly charged small tomated systems under computer control. This discussion
molecules such as inorganic ions, and satisfactory peak will focus on the features of automated CE instrumenta-
tion.
shapes can only be obtained by carefully matching sam-
ple and electrolyte conductivities. The effect is much less
noticeable in other CE separations, except at high sample
A. Power Supply
concentrations.
Power supplies capable of delivering constant voltage with
high precision up to 30 kV are standard throughout the
C. Joule Heating (h joule )
industry. Most systems offer, in addition, constant cur-
When an electric field is applied to a capillary containing rent operation at up to 300 µA; constant current operation
an electrolyte, Joule heat is generated uniformly across may be desirable in systems without adequate tempera-
the circumference of the tube. Since heat can only be re- ture control. High voltage is applied at the capillary inlet,
moved at the margin of the tube, a temperature gradient with the outlet (detector) at ground potential. When sepa-
exists across the radius of the tube. As noted in Eq. (2), rations are performed in uncoated fused silica capillaries
mobility is inversely related to viscosity, which decreases in the presence of EOF, the inlet (high-voltage) electrode
with temperature. Mobility increases approximately 2.5% is the anode and EOF carries analytes towards the cathode
for each degree rise in temperature. Therefore, the temper- (“normal” polarity). Separations performed in coated cap-
ature gradient creates a mobility gradient across the tube illaries without EOF may require “reversed” polarity. In
radius which contributes to band broadening. It should some separations, the direction of EOF may be reversed
also be noted that, in addition to this mobility gradient, by using capillaries coated with positively charged poly-
Joule heat can compromise resolution by increasing dif- mers or by using osmotic flow-modifying additives in the
fusion and convection; these are minimized by the use of background electrolyte. In these cases, reversed polarity
viscous buffers. is also used.