CEhandout - Capillary Electrophoresis Capillary...

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Unformatted text preview: Capillary Electrophoresis Capillary Electrophoresis Introduction In the analytical laboratories all over the world, capillary electrophoresis (CE) has become a major tool for the study of biologically important mixtures. Unlike GC and HPLC, which separate components of a mixture based on their polarity or size, CE employs a potential field to separate positively charged, negatively charged, and neutral species. On the basis of this separation method, the number of theoretical plates (and thus separation efficiency) is much greater with CE than for GC or HPLC. Number of theoretical plates = 2 1/2 m w t 5.54 N = where t m is the migration time and w 1/2 is the peak width at half-height. Because the separation efficiency with CE is so high, complex mixtures can be separated with baseline resolution in a very short time. High efficiency and resolution are two characteristics that make CE an important analytical tool. Shown below is a block diagram of a CE instrument. A sample chamber contains one end of a capillary and one lead from the high voltage power supply. The collection chamber houses the other end of the capillary and also the other high voltage lead. Near the collection end are the detector optics usually consisting of an ultraviolet-visible spectrophotometer, but other detector configurations are available including fluorometry and electrochemical detection. When a potential is applied across the capillary, individual species will move in the capillary by two distinct methods. The first method of movement is based on the characteristics of the species itself and is called electrophoretic mobility . It is important to note that electrophoretic mobility, or ep , is proportional to electric charge on the species and inversely proportional to certain frictional forces. In short, molecules with a positive charge will tend to flow toward the negative terminal, negative species toward the positive terminal, with neutral species having no electrophoretic mobility. The second method of movement of an individual species is based on the bulk flow of the buffer solution. This phenomenon is called electroosmotic flow and is a result of a double-layer that is formed between the negatively charged walls of the capillary and the cations of the buffer. Cations are pulled toward the negative terminal and through viscous forces pull the solution along with them. In many experiments, the electroosmotic mobility, eo , is greater than the electrophoretic mobility, and consequently, all species will be transported from the sample end to the collection chamber with separation being determined by charge and frictional forces. The time it takes for a species to reach the detector is the migration time and is given by: t m = L d L t eo + ep ( ) V where L d is the length of the capillary to the detector, L t is the total length of the capillary, and V is the applied voltage across the capillary....
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CEhandout - Capillary Electrophoresis Capillary...

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