Overview of Capillary Electrophoresis Separation Techniques
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Capillary zone electrophoresis (CZE)
CZE is the most widely-used mode of CE and is used for the separation of both anionic and cationic solutes in a single analysis. In CZE the anions and cations migrate in different directions, but they are all swept towards the cathode because the electroosmotic flow (EOF) is usually significantly higher than the solute velocity due to migration (mobility). In a typical analysis, cations elute first because the direction of migration is the same as the direction of EOF. Neutral solutes elute next, but are unresolved because they have no mobility and move only with the EOF. Anions elute last because they migrate in the opposite direction to the EOF.
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Capillary gel electrophoresis (CGE)
CGE is the CE-analog of traditional slab-gel electrophoresis and is used for the size-based separation of biological macromolecules such as oligonucleotides, DNA restriction fragments and proteins. The separation is performed by filling the capillary with a sieve-like matrix, for example, cross-linked polyacrylamide, agarose or even solutions of linear polymer. The main advantages over slab-gel electrophoresis are a wider range of gel matrix and composition, online detection, improved quantification and automation.
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Capillary isoelectric focusing (CIEF)
CIEF is used to separate biological molecules mainly proteins;based on differences between the isoelectric points (pI). CIEF is performed by filling the capillary with a mixture of ampholytes and the sample, and then forming a pH gradient. By applying an electric field across the capillary with a basic solution at the cathode and an acidic solution at the anode, the ampholytes and solutes migrate until they reach a region where their overall charge is neutral (pH=pI). The ampholyte and solute zones remain extremely narrow because diffusion to a zone of different pH results in the generation of charge and subsequent migration back to the proper zone.
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Micellar electrokinetic chromatography (MEKC)
MEKC is a unique mode of CE because it can separate neutral as well as charged solutes. In MEKC ionic surfactants are added to the running buffer to form micelles. Micelles have a three-dimensional structure with the hydrophobic moieties of the surfactant in the interior and the charged moieties at the exterior. The separation of neutrals is based on the hydrophobic interaction of solutes with the micelles. The stronger the interaction, the longer the solutes migrate with the micelle. The selectivity of MEKC can be controlled by the choice of surfactant and also by the addition of modifiers to the buffer.
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Isotachophoresis (ITP)
ITP uses two different buffer systems. The solutes are sandwiched between leading and trailing electrolytes, creating a steady state in which the solute zones migrate in order of decreasing mobility. Two unique aspects of ITP are that all solute zones migrate at the same velocity and that they all adopt the concentration of the leading electrolyte. The latter aspect makes ITP a very useful technique for the analysis of dilute solutions. Samples can be concentrated to many orders of magnitude.
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Capillary electrochromatography (CEC)
CEC is a fusion of liquid chromatography (LC) and CE. In CEC the capillary is packed with a stationary phase similar to those used in LC. When an electric field is applied the EOF moves the mobile phase through the packed column. A uniform velocity profile is created which minimizes peak dispersion. The selectivity of the separation—as in LC—depends on partition between the stationary and mobile phases. This allows high efficiency separation of neutral compounds within very short analysis times.
CEC has three specific benefits:
- separation of closely related compounds
- shorter run times and increased sample throughput, and
- substitution of gradient HPLC methods by isocratic CEC.
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