Proteomics/Protein Separations- Electrophoresis/Capillary Electrophoresis
Procedure
[edit | edit source]In capillary electrophoresis (CE), electrophoresis is conducted inside small tubes filled with buffer, called narrow-bore capillaries. The capillaries used in the electrophoresis instrument typically have an internal diameter in the range of 25 to 100 μm.
The ends of a capillary are placed in separate buffer solutions and electrodes connected to a high-voltage power supply are placed in the buffers. One of the buffer reservoirs, usually at the anode, is replaced by sample. The sample is introduced into the capillary by capillary action, siphoning, or pressure. When an electric current is passed across the capillary, molecules are separated. Separated analytes are detected directly through the capillary wall by a UV or fluorometric detector connected near the opposite end.[1]
Flow and dispersion
[edit | edit source]In other separation techniques like high pressure liquid chromatography, separations are driven by pressure. That results in frictional forces in places where the mobile phase is in contact with solid surfaces. These frictional forces cause the velocity of mobile phase close to the wall to be zero while the force in the center is large, resulting in a parabolic flow profile in the capillary. This profile results in solute zones being broadened as they move through the capillary, reducing the resolution of the separation. In CE, the flow is flat, which results in much higher resolution than comparative pressure driven techniques.
Visualization
[edit | edit source]The data output from CE is an electropherogram, which is a plot of migration time versus detector response. The response by detector is usually concentration dependent fluorescence or UV-visible absorbance. Different peaks are shown in the electropherogram for separated analytes depending on different retention times. The electropherogram shows separation peaks of cationic, anionic and neutral solutes in the mixture.
Advantages
[edit | edit source]CE is advantageous in many ways. The heating that takes place due to high voltage loads on slab gels can have a negative effect on the separation of the proteins, and the use of capillaries lessens said heat buildup. Also, because the gel will not need to be handled, it can allow one to use liquid polymers for separation, and can be replaced between runs. Automation is also much more possible with this technique, lessening the time it takes, and making reproducible results.
See also
[edit | edit source]The Wikipedia page for capillary electrophoresis can be found here.
Wikipedia details for electropherograms can be found here.
References
[edit | edit source]^ "Capillary Electrophoresis: A Simple Technique." http://www.beckmancoulter.com/resourcecenter/labresources/ce/cedefinitionmodes.asp