Capillary zone electrophoresis characteristics

The application of high-performance capillary zone electrophoresis (HP-CZE) in its various selectivity modes has become a very valuable adjunct to HPLC for the analysis of peptides. For synthetic peptides, in particular, both HPLC and HP-CZE now form essential components of the analytical characterization of these molecules. Increasingly, zonal, micellar, or (biospecific) affinity-based HP-CZE procedures with open tubular capillary systems are adapted to allow resolution with extremely high separation efficiencies (e.g., >105 plates per meter) of synthetic or naturally occurring peptides as part of the determination of their structural, biophysical, or functional properties. Illustrative of these capabilities are the results shown in Figure 19 for the separation of several peptides with different charge and Stokes radius characteristics by HP-CZE. 

Although methods based on capillary zone electrophoresis have not achieved the popularity of HPLC methods, a few studies have been reported. The defining characteristics of these are summarized in Table 5. 

The adsorptive and voltammetric characteristics of Cu(II) complexes with guanine, guanosine and adenosine were exploited [120] in order to detect these bases after separation by capillary zone electrophoresis, and the enzyme-mimic catalytic activity of a DNA-Cu2+ complex [121] was used to develop an amperometric quinacrine sensor using an oxygen electrode covered by the complex entrapped in polyacrylamide gel. 

In practice, both electrophoretic methods have their advantages. Capillary isoelectric focusing enables rapid and precise determination of isoelectric points but provides no information on the general pH-dependence of mobility or pfCa values. In addition, because of its high efficiency cIEF can reveal impurities which are not detectable under the broader peaks of CZE (Fig. 31). Capillary zone electrophoresis, however, determines pi values only indirectly pKa characteristics can also be determined but this takes longer. 

The charge density of biomolecules is related to their electrophoretic mobility therefore, electrophoresis in a free solution capillary zone electrophoresis (CZE)) can be a method to separate biomolecules based on the charge density. The free solution electrophoretic mobility is a characteristic feature of each analyte, which is determined by the complicated balance among the electrical driving force on the analyte, electrical driving force on the counterions within the... 

An entirely different concept in analytical separations is provided by capillary electrophoresis (CE) in which the flow of liquid is generated by electro-osmotic flow (EOF) driven by an external electric field. The major advantage of this approach is the essentiaUy flat plug flow profile that leads to intrinsically more narrow elution peaks than the parabolic flow profiles characteristic of pressure-driven viscous flows. In capillary zone electrophoresis (CZE) separation is achieved by superimposing the different electrophoretic mobihties of the solutes on to the EOF. In electrochromatography the separation is achieved as in packed column HPLC but using an EOF to generate flow of the mobile phase past the stationary phase particles. The importance of these EOF-based techniques is their application to miniaturized devices, lah-on-a-chip or micro total analysis systems. Such devices that can be directly interfaced to a mass spectrometer via an ESI source are currently under intense development. 

CZE is high voltage, free-solution electrophoresis carried out in a capillary. The capillary is filled with the running electrolyte (a buffer solution), and the ionic analytes are separated on the basis of the differences in their electrophoretic mobilities. The favorable ratio of surface area to volume allows the dissipation of the Joule heat from the capillary and the application of high electric fields with rapid and efficient separations. Also, the anticonvective characteristic of the capillary limits the process of zone diffusion, maintaining the efficiency of separation without the need of further anticonvective media such as gels. 

Figure 9.3 depicts the concept of ECEEM schematically. In contrast to NECEEM, in ECEEM the capillary is filled with T at a concentration identical to that in the equilibrium mixture. Therefore, quasiequilibrium will be maintained during electrophoresis if the time reqnired for reeqnilibration is shorter than the characteristic time of separation. In snch a case, the equilibrium mixture will be migrating as a single electrophoretic zone with an effective velocity being a function of fifd and [T], If Kd [T], most of L will be free and the... 

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