Capillary isoelectric focusing principle

Capillary isoelectric focusing separates analytes based on differences in their isoelectric points (pi) using the same principles as in preparative solution IEF. After a focusing step, that builds up a linear pH gradient in the capillary (controlled with zwitterionic internal markers), the analytes move as a function of their respective charge until they reach a position of zero charge (isoelectric point). The solution is then mobilized in CIEF to the detector hydrodynamically. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

The next four chapters discuss the basic principles underlying operation and method development of the most common electrodriven analytical techniques CE, capillary isoelectric focusing (cIEF), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE). Weinberger presents a comprehensive approach for method development in CE with an emphasis on small-molecule applications. This is followed by Kilar s chapter describing the principles of and method development in cIEF, as well as recent innovations... 

Capillary electrophoresis is the generic name for a family of related techniques which have their origin in capillary zone electrophoresis (CZE) and are capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), miscellar electrokinetic capillary chromatography (MECC) and capillary isotachophoresis (CITE). Though the techniques differ significantly in principle of operation they can be carried out largely on the same basic instrumentation. 

Figure 11 (Concentration gradient imaging detection in capillary isoelectric focusing. (A) Experimental arrangement and (B) principle of the imaging system. (C) Images of focused transferrin (iron poor form) and (D) after reaction with ironflll). (Reprinted with permission from Wu J and Pawliszyn J (1992) Analytical Chemistry 64 224-227 and 2934-2941 American Chemical Society.)...

FIGURE 52.7. Separation principle of capillary isoelectric focusing A-F ampholytes and analyzed compounds. 

The most distinguishing phenomenon in capillary electrophoresis is whether the experiments are performed in the absence or in the presence of electroos-motic flow (EOF), (see Chapter 6 for details on EOF). Unlike other types of capillary electrophoresis, isoelectric focusing can be performed under both modes. Since the experimental and theoretical principles governing these modes of CIEF are different, they will be discussed separately. 

A great advance in the equipment for isoelectric focusing has recently been made by Valmet (40). He has described a new principle for electrofocusing called zone convection electrofocusing. In this technique the stabilizing against convection does not call for any capillary system, density gradient or membranes. 

See also-. Capillary Electrophoresis Overview. Electrophoresis Principles Isotachophoresis Isoelectric Focusing Polyacrylamide Gels Two-Dimensional Gels Blotting Techniques. 

See also Blood and Plasma. Capillary Electrophoresis Overview. Cerebrospinal Fluid. Clinical Analysis Sample Handling. Electrophoresis Principles Isoelectric Focusing Polyacrylamide Gels Clinical Applications. Immunoassays Overview. Immunoassays,... 

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