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Capillary electrophoresis is one type of electrophoresis in which the separation of molecules is performed in a capillary tube that is immersed in an electrophoresis running solution. It is the only type of electrophoresis that can be modified to be fully-automated and to accommodate all forms of electrophoresis, this makes CE a powerful and versatile technique.
Principles of Capillary Electrophoresis
Capillary electrophoresis (CE) is an analytical technique that separates molecules in a long and narrow silica-fused tube filled with a solution. Similar to other types of electrophoresis, the separation by CE is initiated when voltage is applied to the system, and the separation is conducted under an electric field, resulting in the difference in the mobility of each molecule.
Under the influence of an electric field, molecules in a solution that possess ionizable functional groups are ionized, turning into either positively or negatively charged ions. The electric force that exists between the two electrodes propels the ions to move towards the opposite charge. The pace at which each ion moves, the electrical mobility, depends on its mass, overall charge and how the ion interacts with other components in the system.[1,2]
In CE, the electrophoretic separation occurs in a capillary tube of about 25-75µm in diameter. A liquid sample containing molecules to be separated is first injected into the capillary either by applying pressure or high voltage power. After injection, the electric field ionizes the molecules in the sample and forces them to migrate from one end of the capillary tube to the other end.
At the detection window, an area in or outside of the capillary, the presence of the ions are detected using, for example, UV-absorbance, fluorescence, laser-induced fluorescence (LIF), or electrochemistry. The differences in the electrical mobility of the ion are reflected in the time it takes to migrate to the detection window, this is termed retention time or migration time and is shown as an electropherogram.
Figure 1: Example of an electropherogram from the sequencing of an unknown DNA fragment by automated capillary electrophoresis.
High Surface-To-Volume in CE Increases the Efficiency in Heat Dissipation
Capillary electrophoresis uses the same principles as other types of electrophoresis and is subjected to Joule heating, the conversion of electrical energy into thermal energy or heat.[1,2] In electrophoresis, it occurs when the current meets the resistance of the components in the system.
The generated electrical energy is subsequently converted into thermal energy, which heats the fluid in the electrophoresis system.[2,3] The heated fluid diffuses from its source and is replaced by the cooler fluid from the other part of the system. The replacing fluid is heated and subsequently displaced by cooler fluid, creating a cycle of convection currents that continue until heat is evenly distributed throughout the system.
Excessive heat can damage heat-sensitive samples, disrupt the separation and destabilize the system. As a result, the application of high power to the system, which can accelerate the ion mobility and decrease the separation time is generally restrained in electrophoresis.
However, in CE, electrophoresis is performed in a capillary tube, this affords the system with a high surface-to-volume environment. Thus, the distribution and subsequent dissipation of thermal energy are more efficient. It enables CE to be performed in a high electrical power field, and it reduces the separation time without having to compensate for the resolution.
Electroendosmosis Flow Adds to The Speed and Versatility Of CE
Apart from the high surface-to-volume ratio, the capillary also affords CE with a moving force that drives the ion’s migration, in addition to the electrical force. On the interior surface of the capillary, the ionized solution causes the surface to be ionized, and the solvated ions are adsorbed onto the interior wall of the capillary. The ions on the stern layer, the interior wall of the capillary, attract the ions of the opposite charge in the solution to build up nearby, forming a diffuse layer. Thus, an electrical double layer is formed at the interface of the surface and the solution.
When voltage is applied, the ions at the diffuse layer are pulled towards the electrode of the opposite charge, creating an electroendosmosis flow, also termed electroosmotic flow (EOF) that tows other solvated ions to the same destination, whether their net charge is the same or opposite. EOF is considered to be a hallmark of CE, which further reduces the separation time and allows all ion species, anions, cations and neutrals, to be separated in a single electrophoresis run.[3,4]
Figure 2: Diagram showing the components of an electrophoresis unit in capillary electrophoresis.