Capillary zone electrophoresis principles

Beckers, J. L. and Bocek, P., Sample stacking in capillary zone electrophoresis Principles, advantages and limitations. Electrophoresis, 21, 2747, 2000. 

Electrodriven Separation Techniques encompass a wide range of analytical procedures based on several distinct physical and chemical principles, usually acting together to perform the requh ed separation. Example of electrophoretic-based techniques includes capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP), and capillary gel electrophoresis (CGE) (45-47). Some other electrodriven separation techniques are based not only on electrophoretic principles but rather on chromatographic principles as well. Examples of the latter are micellar... 

In CZE, the capillary, inlet reservoir, and outlet reservoir are filled with the same electrolyte solution. This solution is variously termed background electrolyte, analysis buffer, or run buffer. In CZE, the sample is injected at the inlet end of the capillary, and components migrate toward the detection point according to their mass-to-charge ratio by the electrophoretic mobility and separations principles outlined in the preceding text. It is the simplest form of CE and the most widely used, particularly for protein separations. CZE is described in Capillary Zone Electrophoresis. ... 

The corresponding equations of USP <727> are included in section Principles of Capillary Zone Electrophoresis. 

Subsequently four different CE modes are described in the sections Capillary Zone Electrophoresis, Capillary Gel Electrophoresis, Capillary Isoelectric Focussing, and Micellar Electrokinetic Chromatography (MEKC), respectively. The fundamental principles of the specific separation modes are briefly explained, using appropriate equations where required. In Table 3 all equations are listed. In addition, the influence of both instrumental parameters and electrolytic solution parameters on the optimization of separations is described. 

Muzikar, J. van de Goor, T. Kenndler, E. The Principle Cause for Lower Plate Numbers in Capillary Zone Electrophoresis with Most Organic Solvents. Anal. Chem. 2002, 74, 434-439. 

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. 

F. Foret, L. Kivdnkovd, and P. Boek, Principles of capillary electrophoretic techniques Micellar electroki- 8. netic chromatography, in Capillary Zone Electrophoresis (B. J. Radola, ed.), VCH, Weinheim, 1993, 9. 

What is the principle of separation by capillary zone electrophoresis ... 

Application of some of the instrumentation principles of HPLC to electrophoresis, bearing in mind the need to cause all of the components of a mixture to migrate differently, has led to the development of several related techniques that are particularly useful in the amino-acid and peptide fields (Baker, 1995). A typical electro-pherogram (Figure 4.16) indicates the salient features of the capillary zone electrophoresis (CZE) analysis protocol. 

In isotachophoresis, electrical detection is most commonly used, although thermal and UV-absorption detection are also used(3). For capillary zone electrophoresis, UV-absorption and fluorescence detection have proven most useful so far. The principles behind the optical detection modes are fairly obvious. However, the electrical and thermal detectors deserve further explanation. As described earlier, in isotachophoresis, each zone is an individual "pure" band of sample ions. The zones travel in order of decreasing mobility. To compensate for each successive... 

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. 

Capillary zone electrophoresis. The technique of CZE was first introduced by Jorgenson [86]. Operating principles have already been... 

Other Techniques. A growing technique related to lc/ms and regarded as complementary to it is that of capillary zone electrophoresis/mass spectrometry (cze/ms) (22). Using cze/ms, high resolution separation of water-soluble compounds is accompHshed by the principles of electrophoresis (qv). The sample is then coupled to the mass spectrometer by electrospray ionization (23) or a fast atom bombardment interface (fab) to produce molecular ions (24). Biotechnology applications of cze/ms have great potential (25). 

Similarly to gel electrophoresis, a number of modes can be employed that separate analyte mixtures according to different properties. Some of these modes are summarised in Table 3.2 together with their commonly used abbreviations. Depending on the principle of separation, different species can be analysed. After an overview of capillary electrophoretic instrumentation, the different modes of capillary electrophoresis are described in more detail including capillary zone electrophoresis (CZE), capillary isoelectric focussing (CIEF), micellar electro-kinetic chromatography (MEKC) and capillary gel electrophoresis (CGE). 

Capillary zone electrophoresis is not only the simplest form of CE, but also the most commonly utilized. Discussion of this mode permits the presentation of a generic design for the instrumentation for CE. The addition of specialized reagents to the separation buffer readily allows the same instrumentation to be used with the other modes mentioned in the previous section addition of surfactants with MEKC, ampholines for CIEF and a sieving matrix (linear polymers, entangled matrices) for CGE. The discussion on CZE in the following subsections allows for analysis of some of the basic principles governing analyte separation by this technique. 

With CZE, the normal polarity is considered to be [inlet—(+), detector—(—) outlet] as shown in Figures 1.4 and 1.5. As electrophoresis ensues, the analytes separate according to their individual electrophoretic mobilities and pass the detector as analyte zones (hence, the term capillary zone electrophoresis or CZE). The fact that, under appropriate conditions, all species (net positive, net negative, or neutral) pass the detector indicates that a force other than electrophoretic mobility is involved. If the applied field were the only force acting on the ions, net positively charged (cationic) substances would pass the detector while neutral components would remain static (i.e., at the inlet) and anionic components would be driven away from the detector. It is clear that, if this were the case, CE would be of limited use. Fortuitously, there is another force, electroosmotic flow (EOF), driving the movement of all components in the capillary towards the detector when under an applied field (and a normal polarity). EOF plays a principle role in many of the modes of CE and most certainly in CZE. This is discussed briefly in the next section. 

Figure 3 Principle of capillary zone electrophoresis. The EOF leads to the electroosmotic movement of all compounds towards the cathode. However, electrophoretically the anions migrate towards the anode, the higher charged the faster. If the electrophoretic velocity is superior to the electroosmosis, these anions would not be detected. Neutral compounds...

Foret, F. Kivankova, L. Boek, P. Principles of capillary electrophoretic techniques Micellar electrokinetic chromatography. In Capillary Zone Electrophoresis, Radola, B.J., Ed. VCH Weinheim, 1993 67-74. 

Owing to its outstanding high-performance separation potential also capillary electrophoresis has been employed in different modes and with different detection methods for separation and determination of MC. Both capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), which employs a surfactant above the critical micelle concentration to separate neutral and uncharged molecules based on electromigration principle, have been employed for determination of... 

Micellar electrokinetic chromatography performed in capillaries is a separation technique combining some of the operational principles of micellar liquid chromatography and capillary zone electrophoresis. This technique was termed micellar electrokinetic capillary chromatography (MECC) by Burton et al. [79]. MECC uses the addition of a surface-active agent in the working electrolyte, which creates new possibilities for electrophetic separations. 

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