It consists of a high-voltage power supply, two buffer reservoirs, a
capillary and a detector. This basic set-up can be elaborated upon with enhanced features
such as autosamplers, multiple injection devices, sample/capillary temperature control,
programmable power supply, multiple detectors, fraction collection and computer
interfacing. Different modes of capillary electrophoretic separations can be performed
using a standard CE instrument. The origins of the different modes of separation may be
attributed to the fact that capillary electrophoresis has developed from a combination of
many electrophoresis and chromatographic techniques. In general terms, it can be
considered as the electrophoretic separation of a number of substances inside of a narrow
tube. Even though most applications have been performed using liquids as the separation
media, capillary electrophoretic techniques encompass separations in which the capillary
contains electrophoretic gels, chromatographic packing or coating.
The distinct capillary electroseparation methods
include:
Currently CZE is the most commonly used technique in CE. Many
compounds can be separated rapidly and easily. The separation in CZE is based on the
differences in the electrophoretic mobilities resulting in different velocities of
migration of ionic species in the electrophoretic buffer contained in the capillary.
The separation mechanism is mainly based on differences in solute size and charge
at a given pH. The electroosmotic flow in uncoated fused silica capillaries is usually
significant with most commonly used buffers. It is also significantly greater than the
electrophoretic mobility of the individual ions in the injected sample. Consequently both
anions and cations can be separated in the same run. Cations are attracted towards the
cathode and their speed is augmented by the electroosmotic flow. Anions, although
electrophoretically attracted towards the anode, are swept towards the cathode with the
bulk flow of the electrophoretic medium. Under these conditions, cations with the highest
charge/mass ratio migrate first, followed by cations with reduced ratios. All the
unresolved neutral components are then migrated as their charge/mass ratio is zero.
Finally, the anions migrate. Anions with lower charge/mass ratio migrate earlier than
those with greater charge/mass ratio. The anions with the greatest electrophoretic
mobilities migrate last. One important point to note is that it is possible to change the
charge/mass ratio of many ions by adjusting the pH of the buffer medium to affect their
ionization and hence electrophoretic mobility.
The main separation mechanism in CGE is based on differences in
solute size as analytes migrate through the pores of the gel-filled column. Gels are
potentially useful for electrophoretic separations mainly because they permit separation
based on 'molecular sieving'. They serve as anti-convective media, minimize solute
diffusion, which contributes to zone broadening, prevent solute adsorption to the
capillary walls and they help to eliminate electroosmosis.
The main separation mechanism is based on solute partitioning
between the micellar phase and the solution phase. The technique provides a way to resolve
neutral molecules as well as charged molecules by CE. Micelles form in solution when a
surfactant is added to water in concentration above its critical micelle concentration
(CMC). Even though these anionic micelles are attracted toward the anode, in an uncoated
fused silica capillary they still migrate toward the cathode because of electroosmotic
flow. However, the micelles move toward the cathode at a slower rate than the bulk of the
liquid because of their attraction towards the anode. Neutral molecules partition in and
out of the micelles based on the hydrophobicity of each analyte. Consequently the micelles
of MEKC are often referred to as pseudo (or moving) stationary phase. A very hydrophilic
neutral molecule, eg methanol, will spend almost no time inside the micelle and will
therefore migrate essentially at the same rate as the bulk flow and elute earlier. On the
other hand, a very hydrophobic neutral molecule eg Sudan III, will spend nearly all the
time inside the micelles and will therefore elute later, together with the micelles. All
other solutes with intermediate hydrophobicity will migrate within this migration window.
MEKC can be used with ionic substances as well as neutral compounds.' A combination of
charge/mass ratios, hydrophobicity and charge interactions at the surface of the micelles
combine to affect the separation of the analytes.
The main feature of CITP is that it is performed in a discontinuous
buffer system. Sample components condense between leading and terminating constituents,
producing a steady-state migrating configuration composed of conservative sample zones.
This mode of operation is therefore different from other modes of capillary
electrophoresis, eg CZE, which are normally carried out in a uniform carrier buffer and is
characterized by sample zones which continuously change shape and relative position. In
the case of a typical CZE separation, the electropherogram obtained contains sample peaks
similar to those obtained in chromatographic separations, whereas in the case of CITP, the
isotachopherogram obtained contains a series of steps, with each step representing an
analyte zone. Unlike in other CE modes, where the amount of sample present can be
determined from the area under the peaks as in chromatography, quantitation in CITP is
mainly based on the measured zone length which is proportional to the amount of sample
present.
In CIEF, substances are separated on the basis of their isoelectric
points (pH values). The most common type of sample that utilizes this analytical method is
protein. The protein samples and a solution that forms a pH gradient are placed inside a
capillary. The anodic end of the column is placed into an acidic solution (anolyte), and
the cathodic end in a basic solution (catholyte). Under the influence of an applied
electric field, charged proteins migrate through the medium until they reside in a region
of the pH where they become electrically neutral and therefore stop migrating.
Consequently, zones are focused until a steady state condition is reached. After focusing,
the zones can be migrated (mobilized) from the capillary by a pressurized flow.
Alternatively, after focusing, salt (e.g. NaCl) can be added to the anolyte (acid
reservoir) or catholyte. By the principle of electroneutrality, Na+ can exchange for H+ in
the tube, generating a pH imbalance gradient which causes the migration of the components.
Sharp peaks are obtained with good resolution, and a large peak capacity is observed
mainly because the whole tube is simultaneously used for focusing.
