Technique of Zone Electrophoresis

Recently, detailed accounts of the methodology of zone electrophoresis have appeared, so that only the salient points of the technique need be mentioned here. 

In view of the fact that filter paper is the supporting matrix by far the most frequently used in zone electrophoresis, only those types of apparatus which employ filter-paper strips will be mentioned. 

in zone electrophoresis, the impregnated, filter-paper strip is the effective resistance in an electrical circuit, heat will be generated within its structure. Ineffective or incomplete dissipation of this heat will lead consecutively to evaporation of the conducting solution, establishment of a concentration gradient, and, ultimately, the breakdown of the circuit if a dry zone is formed across the paper strip. The various types of apparatus in current use may be classified according to the devices employed to control the development of heat within the paper strip or to facihtate its dissipation. 

A convenient procedure for preparation of the paper strip is as follows. 

Several strips may be used simultaneously if they are arranged in sandwich fashion and are separated by polythene sheets. The number of papers which may be so used will be limited by the amount of heat to be dissipated, which, in turn, will depend upon the thickness of the paper strips, the ionic strength of the conducting solution, and the potential gradient apphed. 

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In CE, the classic techniques of zone electrophoresis, ITP, lEF, and gel electrophoresis are carried out in a small-bore (10- to 100- im internal diameter) fused silica capillary tube of 20 to 200 cm in length. ... 

CE combines the classic techniques of zone electrophoresis, isotachophoresis, isoelectric focusing, and gel electrophore-... 

A number of years later. Smithies Introduced starch gels in the 1950 s (, ). His technique of zone electrophoresis with starch gel as the supporting medium gave a resolving power equal, if not greater, to that of the Tiselius method. Shortly thereafter,... 

GENERAL TECHNIQUES OF ZONE ELECTROPHORESIS 1. PAPER ELECTROPHORESIS... 

Zone electrophoresis is influenced by adsorption and capillarity, as well as by electroosmosis. Therefore evaluation of mobility (and f) from this type of measurement is considerably more complex than from either microelectrophoresis or moving-boundary electrophoresis. Nevertheless, zone electrophoresis is an important technique that is widely used in biochemistry and clinical chemistry. One particularly important area of application is the field of immunoelectrophoresis, which is described briefly in Section 12.11. Additional information on zone electrophoresis may be obtained from Probstein (1994) and Hunter (1981) and the references given there. Variants of zone electrophoresis also exist see, for example, Gordon et al. (1988) for information on a variant known as capillary zone electrophoresis and Righetti (1983) for information on what is known as isoelectric focusing. 

V7. von Frijtag, C. A. J., and Reinhold, ]. G., Application of zone electrophoresis to analysis of serum proteins. Technique for horizontal strip method and evaluation of its precision and accuracy. Anal. Chem. 27, 1090 (1955). 

The two-dimensional techniques, both as a preparative tool for collecting fractions and perhaps especially as a separative tool in rapid star electrophoresis, will undoubtedly prove to be of great value in clinical laboratory work. The recent technical improvements in the apparatus allow for easy working conditions. These were previously lacking and delayed the clinical use of methods which are based on a different principle from that of zone electrophoresis. They yield important results in fields where the one-dimensional method has partly failed. 

Capillary electrophoresis is a general term that is used to describe a number of different separation techniques. Capillary zone electrophoresis (CZE) is the classic technique and is therefore usually referred to as just CE. Other techniques include micellar electrokinetic chromatography (MEKC), capillary isoelectric focusing, and capillary isotachophoresis. CZE and MEKC are the predominant techniques and are those used herein, so only they will be discussed in detail here. 

By a suitable combination of some fundamental electrophoretic techniques their advantages can be exploited for better separations. For instance, in discontinuous electrophoresis (disc-electrophoresis), isotachophoretic arrangement is utilized in the first part of the experiment in order to concentrate the sample components, and to arrange them according to their effective mobilities. In the second part of the experiment individual zones are separated on the principles of zone electrophoresis. In other cases combinations of electromigration and other principles (for instance, immunoelectrophoresis) are exploited. 

This procedure represents a preparative version of zone electrophoresis. The apparatus and technique has been previously elaborated [294,295] separation itself is carried out either in a stream of electrolytic solution or on a sheet of cardboard (curtain electrophoresis). In the free flow version the separation is carried out in a cell formed by two parallel glass plates (50 x 50 cm) situated 0.5-1.0 mm apart. It is necessary to ensure an equal and laminar flow of the electrolyte, which is carried out by feeding the buffer through a multichannel peristaltic pump. The sample is continuously applied in the middle near the upper edge of the cuvette (or paper sheet). The electrophoretic separation occurs transversally between vertical electrodes located on the right and left hand side of the separation cuvette (Fig. 6.33). The separated fractions are collected at the lower end of the cell by a system of small communicating vessels or by a multi-channel pump. In the version using paper the... 

Microchip capillary electrophoresis (MCE), like conventional capillary electrophoresis (CE) and most other chemical separation techniques, is heavily dependent on surface chemistry. This connection is obvious for techniques that use a stationary phase but is also apparent for separation modes like zone electrophoresis where the separation is based on the mobility of the ions. An important component of zone electrophoresis is the electroosmotic flow (EOF), which is a surface-derived phenomenon. Furthermore, the degree of surface hydrophobicity can cause adsorption resulting in band broadening. As a result of the significance of the surface chemistry, it is important to develop an understanding of methods that provide control of surface chemistry in MCE. This problem is more... 

It seems that unless the test material is relatively pure (from a preceding purification step if needed) NMR will be used in more of a structure confirmation role than for first identification. Two relatively new applications of chromatographic techniques. Capillary Zone Electrophoresis (CZE) and High Performance Liquid Chromatography (HPLC), to the analysis of CW materials generated great interest. 

Biomolecule Separations. Advances in chemical separation techniques such as capillary zone electrophoresis (cze) and sedimentation field flow fractionation (sfff) allow for the isolation of nanogram quantities of amino acids and proteins, as weU as the characterization of large biomolecules (63—68) (see Biopolymers, analytical techniques). The two aforementioned techniques, as weU as chromatography and centrifugation, ate all based upon the differential migration of materials. Trends in the area of separations are toward the manipulation of smaller sample volumes, more rapid purification and analysis of materials, higher resolution of complex mixtures, milder conditions, and higher recovery (69). 

Disc Electrophoresis. Resolution in zone electrophoresis depends critically on getting sample components to migrate in a focused band, thus some techniques ate employed to concentrate the sample as it migrates through the gel. The most common technique is referred to as discontinuous pH or disc electrophoresis. Disc electrophoresis employs a two-gel system, where the properties of the two gels are different. 

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... 

Zone electrophoresis is defined as the differential migration of a molecule having a net charge through a medium under the influence of an electric field (1). This technique was first used in the 1930s, when it was discovered that moving boundary electrophoresis yielded incomplete separations of analytes (2). The separations were incomplete due to Joule heating within the system, which caused convection which was detrimental to the separation. 

Figure 50-2. Technique of cellulose acetate zone electrophoresis. A A small amount of serum or other fluid is applied to a cellulose acetate strip. B Electrophoresis of sample in electrolyte buffer is performed.

Capillary electrophoresis (CE) has several unique advantages compared to HPLC, snch as higher efficiency dne to non-parabolic fronting, shorter analytical time, prodnction of no or much smaller amounts of organic solvents, and lower cost for capillary zone electrophoresis (CZE) and fused-silica capillary techniques. However, in CZE, the most popular separation mode for CE, the analytes are separated on the basis of differences in charge and molecular sizes, and therefore neutral compounds snch as carotenoids do not migrate and all co-elute with the electro-osmotic flow. 

CE was recently used for anthocyanin analysis because of its excellent resolution. This technique has different modes capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEE), and capillary isotachophoresis (CITP)."° CZE is the most popular method for anthocyanin... 

Very recently, HPLC with fluorescence detection was recommended for improving detection sensitivities of betalains. " While this technique may be worthwhile for betaxanthin analyses, its use for betacyanins cannot be recommended. Although this technique represents a worthwhile approach requiring low amounts of solvent and sample and generally characterized by a high separation efhciency, only one study dealt with the use of capillary zone electrophoresis for betalain analyses. ... 

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