Capillary electrophoresis is a technique that uses very narrow-bore capillaries, typically 50 µm internal diameter and 300 µm external diameter, to separate a wide array of large and small molecules. Figure 1 is a schematic of a CE system. It includes a power supply, capillary, buffer reservoirs, and detector.
Figure 1: Schematic of a Capillary Electrophoresis (CE) system
The heart of the CE system is the capillary where separation takes place. The capillaries are mostly open tubular and not packed, resulting to excellent resolution and very sharp peaks. The high voltage is required to move the buffer and analyte molecules through the capillary. Charged molecules are separated along the capillary due to electrophoretic migration and electroosmotic flow. Electrophoretic migration causes charged molecules to move towards the electrode of opposite charge, hence positive and negative charged molecules migrate at different rates. Electroosmotic flow is due to the presence of charged groups on the surface of the support medium, for instance silanol groups on the surface of the glass wall used in CE.
In CE, the sample is introduced by immersing the end of the capillary into a sample vial and applying pressure, vacuum or voltage. Different modes of capillary electrophoretic separations can be performed using a standard CE instrument, depending on the types of capillary and electrolytes used. Some examples are:
Capillary Zone Electrophoresis (CZE), also known as free-solution CE (FSCE), is the simplest and most widely used form of CE. The separation mechanism is based on differences in the charge-to-mass ratio of the analytes.
Capillary Gel Electrophoresis (CGE) is the traditional gel electrophoresis that takes place in a capillary. It uses polymers in solution to create a molecular sieve that allows analytes having similar charge-to-mass ratios to be resolved by size.
Capillary Isoelectric Focusing (CIEF) separates amphoteric molecules in a pH gradient generated between the cathode and anode. The analyte molecules migrate until it reaches its isoelectric point (pI); migration then stops and the sample is focused into a tight zone.
Micellar Electrokinetic Capillary Chromatography (MECC OR MEKC) is a form of CE in which surfactants are added to the buffer solution at concentrations that form micelles. Separation takes placed because of the differential partition between the micelle and the solvent.
Analytes are detected using one of several possible detection methods - UV-Vis, fluorescence, mass spectrometry, and electrochemical detection.