Page 74 - Academic Press Encyclopedia of Physical Science and Technology 3rd Analytical Chemistry

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

360 Capillary Zone Electrophoresis

B. Injection injection) or application of hydrostatic pressure by ele-
vating the capillary inlet relative to the capillary outlet
Sample injection in CE requires the introduction of very
(gravity injection). Gravity injection has been reported to
small amounts of analyte at the capillary inlet with high
provide reproducible injection of very small sample zones.
precision. All commercial instruments offer electromi-
On the other hand, pressure injection can allow the flex-
gration injection and at least one type of displacement
ibility of introducing larger sample zones; this can be an
injection.
advantage when using injection pneumatics to introduce
Electromigration is the simplest injection method in
a chiral selector or to perform on-column concentration.
CE; the capillary inlet is immersed in the sample solu-
Moderateinjectionpressurescanbeanadvantagewhenin-
tion and high voltage is applied for a brief period (typi-
jecting samples into capillaries containing analysis buffers
cally a few seconds). If no electroosmotic flow is present,
with viscous agents such as sieving polymers.
sample ions enter the capillary by electophoretic mobility
alone; this mode of sample introduction is termed elec-
trophoretic injection. If EOF is present, sample ions will C. Capillary Temperature Control
be introduced by a combination of electrophoretic mobil-
Temperature control of the capillary environment is essen-
ity and electroosmotic flow; this mode is generally termed
tial for attaining satisfactory reproducibility. Inadequate
electrokinetic injection.
temperature control results in variable migration times. In
Electrophoretic injection offers two advantages. First,
CE, peak area depends upon the residence time of the com-
if electrophoretic injection is performed in the absence of
ponent in the detector light path and therefore is dependent
EOF, only species of like charge will enter the capillary.
upon migration velocity. If migration times vary because
This enables discrimination against compounds of oppo-
ofinadequatetemperaturecontrol,peakareaprecisionwill
site charge, simplifying the separation problem. Second,
be poor. Control of capillary temperature above ambient
zone sharpening can be achieved using the stacking princi- temperature may be desirable in special applications—
ple described above. Unfortunately, these advantages are
for example, in performing kinetic studies or on-column
countered by two major limitations. Since sample ions en-
enzyme assays, in the study of protein folding, or for mu-
ter the capillary based on mobility, low-mobility ions will
tation detection in amplified gene sequences. Operation
be loaded to a lesser extent than high-mobility ions. More
at higher temperatures in capillary zone electrophoresis
importantly, presence of non-analyte ions in the sample
will decrease analysis time and may improve peak shape.
will reduce injection efficiency, so electrophoretic injec-
Operation at lower temperatures generally has little ad-
tion is very sensitive to the presence of salts or buffers in
vantage in most modes of capillary electrophoresis.
the sample matrix. The disadvantages of electrophoretic
Capillary temperature control can be achieved by forced
injection argue against its use in routine analysis except in
air or nitrogen convection or by a circulating liquid cool-
cases where displacement injection is not possible (e.g., in
ant. Forced air control permits the use of free-hanging cap-
capillary gel electrophoresis, CGE). Electrokinetic injec-
illaries but is less efficient. Liquid cooling requires that the
tion suffers from the further disadvantage that many sam-
capillarybeenclosedinasealedcartridge,butthecartridge
ple matrices contain components such as proteins which
format provides for automatic alignment of the capillary
adsorb to the capillary wall and change the magnitude of
in the detector light path and reduces time required for
EOF.
capillary installation when changing methods.
Displacement or hydrodynamic injection is usually the
preferred method since analyte ions are present in the sam-
D. Detectors
ple zone in proportion to their concentration in the bulk
sample, and injection efficiency is less sensitive to varia- Most of the detection modes used in HPLC have been
tions in sample ionic strength. However, it should be noted demonstrated for capillary electrophoresis (absorbance,
that the presence of high salt can affect detector response fluorescence, conductivity, electrochemical, radioactiv-
with displacement injection, and variations in the sample ity, mass spectrometry, postcolumn reaction). However,
viscosity (due to temperature variations or the presence of of these, only absorbance, fluorescence, conductivity,
viscosity-modifying components) can affect displacement and mass spectrometry are available for automated CE
injection efficiency. systems.
Two modes of displacement injection have been em-
ployed in commercial CE instruments: application of pos-
1. Absorbance
itive pressure at the capillary inlet and application of vac-
uum at the capillary outlet. The former method can employ As in HPLC, absorbance detection is used in the vast ma-
pressurization of the sample headspace by gas (pressure jority of CE applications, and all commercial CE systems