Capillary electrophoresis (CE) is a family of related separation techniques that use narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. The National Centre for Biomedical Engineering Sciences at NUI, Galway has recently acquired and installed a Beckman-Coulter Methods Development CE system. The system includes a P/ACE MDQ configured with both photodiode array and laser-induced fluorescence detectors, temperature-controlled sample storage and capillary, and automated injection and separation of samples
In CE high voltages are used to separate molecules based on differences in charge and size, as depicted in the figures at left. Injection into the capillary is accomplished by immersing the end of the capillary into a sample vial and applying pressure, vacuum or voltage. Depending on the types of capillary and buffers, CE can be segmented into several separation techniques, detailed in the next sections.
CAPILLARY ZONE ELECTROPHORESIS (CZE) is the simplest form of CE. The separation mechanism is based on differences in the charge-to-mass ratio of the analytes. When the voltage is applied, the anion (-) and cation (+) move in a different direction. The anion (-) electrophoretic migration will be toward the anode and cation (+) electrophoretic migration will be toward the cathode. The endoosmotic flow or bulk flow will be in the direction of the cathode (see figure above). Because of endoosmotic flow both cation and anion migrate toward the cathode. CAPILLARY GEL ELECTROPHORESIS (CGE) is the adaptation of traditional gel electrophoresis into the capillary using polymers in solution as a molecular sieve. This allows solutes having similar charge-to-mass ratios to be resolved by size. CGE is used for the separation of biological macromolecules such as DNA restriction fragments and proteins. The separation is performed by filling the capillary with a matrix, for example, cross-linked polyacrylamide, agarose or even solutions of linear polymer, such as that shown in the figure at right for the separation of HIV RNA hydridised with DNA probe. The main advantages over slab-gel electrophoresis are a wider range of gel matrix and composition, online detection, improved quantification (no need for PCR) and automation.
CAPILLARY ISOELECTRIC FOCUSING (CIEF) allows amphoteric molecules, such as proteins, to be separated by electrophoresis in a pH gradient generated between the cathode and anode. A solute will migrate to a point where its net charge is zero. At this isoelectric point (the soluteأ¢â‚¬â„¢s pI), migration stops and the sample is focused into a tight zone. The example shown at right is for the determination of the pIأ¢â‚¬â„¢s for single amino acid variants of recombinant human monoclonal antibodies.
MICELLAR ELECTROKINETIC CAPILLARY CHROMATOGRAPHY (MEKC) is a mode of electrokinetic chromatography in which surfactants are added to the buffer solution at concentrations which form micelles. MEKC is a unique mode of CE because it can separate neutral as well as charged solutes. 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.The separation principle of MEKC is based on a differential partition between the micelle and the solvent. CHIRAL ANALYSIS: Capillary electrophoresis has proven to be the most ideal analytical tool to assess the purity of enantiomers. The use of modified cyclodextrins, such as the sulfated CDأ¢â‚¬â„¢s pictured at right, for differential host-guest complexation of enantiomers is by far the most common chiral selector. The separation of enantiomers of amphetamine is shown at right.
CAPILLARY ELECTROCHROMATOGRAPHY (CEC) is a hybrid separation method that couples the high separation efficiency of CZE with HPLC, and uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed. Since there is minimal back-pressure, it is possible to use small-diameter packings and achieve very high efficiencies. The example shown above right is the separation of polyaromatic hydrocarbons in a packed silica capillary.
