High-voltage capillary electrophoresis

Hntterer, K. M., Ultra high voltage capillary electrophoresis. Doctorial Thesis, University of North Carolina, Chapel Hill, NC, 2000. 

Fujiwara, S. and Honda, S., Effect of addition of organic solvent on the separation of positional isomers in high-voltage capillary zone electrophoresis, Anal. Chem., 59, 487, 1987. 

Altria, K. D. and Simpson, C. F., Analysis of some pharmaceuticals by high voltage capillary zone electrophoresis, Pharm. Biomed. Anal., 6, 801, 1988. 

Hutterer, K.M., Jorgenson, J.W., Ultra-high voltage capillary zone electrophoresis. Anal Chem. 1999, 71, 1293. 

Electrophoresis has emerged recently as a promising method for the determination of purines, pyrimidines, nucleosides, and nucleotides. Two techniques are used high-voltage paper electrophoresis (PE) and capillary electrophoresis (CE). 

Altria, K.D. Simpson, C.F. High voltage capillary zone electrophoresis Operating parameter effects upon electro-endosmotic flows and electrophoretic mohihties. Chromatographia 1987, 24, 527-532. 

Fig. 1. Capillary zone el trophoresm (CZE). (a) EOF and order of solute migration, (b) Separation of somp , sweeteners and preservatives by CZE capillary, 65 cm. 50 mm Id. buffer, 0.02 M borate, pH 9.4- temolr r voltage, 30 kV, Injection, hydrodynamic so mbar sec detection, UVabsorbance at 192 nm. Reproduced from D m i-i High Performance Capillary Electrophoresis, 1992, with permission from Agilent Technologies UK Ltd and D l i Heijdl ...

Capillary Electrophoresis. Capillaries were first appHed as a support medium for electrophoresis in the early 1980s (44,45). The glass capillaries used are typically 20 to 200 p.m in diameter (46), may be filled with buffer or gel, and are frequendy coated on the inside. Capillaries are used because of the high surface-to-volume ratio which allows high voltages without heating effects. The only limitations associated with capillaries are limits of detection and clearance of sample components. 

A schematic representation of a CE system is presented in Figure 9.1. In this diagram, the CE components have obvious counterparts to those found in slab gel electrophoresis. Instead of buffer tanks there are two small buffer reservoirs, and the capillary takes the place of the gel (or more accurately, a gel lane). The capillary is immersed in the electrolyte-filled reservoirs, which also make contact with the electrodes connected to a high-voltage power supply. A new feature to the conventional gel electrophoresis format is the presence of an online detection system. 

Capillary electrophoresis (CE) instrument is quite simple. A CE, at its core, is merely a high-voltage power supply (capable of voltages in excess of 30,000 V), capillary (approximately 25 to 100 pm inner diameter), buffers to complete the circuit (e.g., citrate, phosphate, acetate, etc.), and a detector (e.g., UV-Vis). There are additional complexities, of course, but at its heart, the CE is a simple instrument. 

To perform capillary electrophoresis the following basic components are necessary a high voltage power source, a detector and a buffer filled capillary (see Fig. 1). 

Since all electrophoretic mobility values are proportional to the reciprocal viscosity of the buffer, as derived in Chapter 1, the experimental mobility values n must be normalized to the same buffer viscosity to eliminate all other influences on the experimental data besides the association equilibrium. Some commercial capillary zone electrophoresis (CZE) instruments allow the application of a constant pressure to the capillary. With such an instrument the viscosity of the buffer can be determined by injecting a neutral marker into the buffer and then calculating the viscosity from the time that the marker needs to travel through the capillary at a set pressure. During this experiment the high voltage is switched off. 

Capillary electrophoresis (CE) is the most rapidly expanding separation technique in pharmaceutical analysis and is a rival to HPLC in its general applicability. The instrumentation is quite straightforward, apart from the high voltages required, but the parameters involved in optimising the technique to produce separation are more complex than those involved in HPLC. The technique is preferred to HPLC where highly. selective separation is required. 

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