Electrophoresis moving boundary

An alternative to particle electrophoresis is moving-boundary electrophoresis. The technique is used to study the movement of a boundary formed between a colloidal sol or solution and the pure dispersion medium under the influence of the electric field. The technique has found some application for determining not only electrophoretic mobility, but also for small-scale separation of species from a mixture for further identification. It found early application in the study of proteins and other dissolved marcomolecules. 

If a protein solution contains a number of different species of different charge characteristics (and therefore different mobilities in a given charge field), the technique may be able to separate the fractions, or at least the peaks, sufficiently to indicate the number of distinct species present. The technique has largely been displaced by more sensitive, and experimentally easy, techniques however, the inexpensive nature of the process still carries some weight in choosing an approach for some apphcations. 

The principle of this technique can be explained on the following example. Let us assume that there are three anions (A, B, C) sandwiched between anode and cathode. In the front, three zones will be formed after introducing the current, namely A, (A, B) and (A, B, C). Also at the other end there will be three zones, namely C, (B, C) and (A, B, C) (Fig. 6.9). The boundaries between the individual zones are determined by the concentration and effective mobilities of the sample constituents, the choice of the anodic and cathodic buffer and to a considerably lesser extent by viscosity, the nature of the solvent and temperature. The boundaries seen in both sides of the separation can be either zone or isotachophoretic boundaries. Thus, e.g., if the anion buffer has a lower electrophoretic mobility than the most 

In comparison to other electrophoretic techniques moving boundary electrophoresis has only Umited applications and can be used for testing, among other things, purity of substances. Its advantage is based on the fact that rather simple equipment can be used (in comparison with isotachophoretic apparatus) zone boundaries are detected with a conductivity and a photometric detector and the whole separation is carried in narrow bore tubes. 

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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. 

Douglas et al. [98] have measured protein (serum albumin, ovalbumin, and hemoglobin) mobilities over a range of pH values using a free-flow electrophoresis apparatus and a particle electrophoresis apparatus. They found good agreement between the two measurements however, they also found some differences between their measurements and those reported in the older literature. They attributed the differences to the use of moving-boundary electrophoresis methods in the early experimental work and to differences in... 

Chrambach, A, Unified View of Moving Boundary Electrophoresis Practical Implications, Journal of Chromatography 320, 1, 1985. 

Influence of the Surface Concentration of BSA. Compared to the corrected moving boundary electrophoretic mobility of BSA in solution, the mobility of BSA adsorbed onto glass is considerably faster at all ionic strengths at 1.96 pg/cm2 and somewhat faster at lower ionic strengths 1.38 pg/cm2. However, at lower adsorption densities (1.05 and 0.64 pg/cm2), the adsorbed BSA moves more slowly in the applied electric field than BSA in moving boundary electrophoresis under otherwise identical conditions, and at the lowest surface adsorption (0.64 pg/cm2) the mobility of the adsorbed BSA are even somewhat slower than in cellulose acetate gel at all conditions of ionic strength investigated. 

TABLE 4 Comparison of the adsorbed BSA electrophoresis results of Tables 1 and 2, with cellulose acetate electrophoresis (Table 3) and moving boundary electrophoresis [6], extrapolated to 30°C (as in Tables 1 and 2) and to the appropriate ionic strengths (Tables 1-3)... 

With the study of the migration of hydrogenium ions (H ) in a phenolphthalein gel by Lodge in 1886 and the description of the migration of ions in saline solutions by Kohlraush in 1897, a basis was set for the development of a new separation technique that we know today as electrophoresis. Indeed, several authors applied the concepts introduced by Lodge and Kohlraush in their methods and when Arne Tiselius reported the separation of different serum proteins in 1937, the approach called electrophoresis was recognized as a potential analytical technique. Tiselius received the Nobel Prize in Chemistry for the introduction of the method called moving boundary electrophoresis. ... 

Also known as copper blue. maunt on blu ) moving-boundary electrophoresis analychem A U-tube variation of electrophoresis analysis that uses buffered solution so that all ions of a given species move at the same rate to maintain a sharp, moving front (boundary). miivii) baun-dre i lek-tro-fo re-sos ... 

Following the above, we address some of the experimental aspects of electrophoresis. In this context, a few other forms of electrophoresis (e.g., moving boundary electrophoresis and zone electrophoresis) are described briefly. The last of these is used when separation of charged species, rather than the measurement of mobilities, is the item of interest (Section 12.9). 

Of the electrokinetic phenomena we have considered, electrophoresis is by far the most important. Until now our discussion of experimental techniques of electrophoresis has been limited to a brief description of microelectrophoresis, which is easily visualized and has provided sufficient background for our considerations to this point. Microelectrophoresis itself is subject to some complications that can be discussed now that we have some background in the general area of electrical transport phenomena. In addition, the methods of moving-boundary electrophoresis and zone electrophoresis are sufficiently important to warrant at least brief summaries. 

Moving-boundary electrophoresis is most widely applied to protein mixtures. In such a case each molecular species travels with a characteristic velocity. After sufficient time the various components in a mixture become effectively separated, and the percentage of each may be determined by measuring the areas under the schlieren peaks. Figure 12.12a shows a... 

FIG. 12.11 Schematic illustration of a Tiselius-type moving boundary electrophoresis apparatus. 

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. 

What are moving-boundary electrophoresis and zone electrophoresis Where are they used ... 

Longsworth, L. G., Moving Boundary Electrophoresis - Practice. In Electrophoresis, Vol. 1 (M. Bier, Ed.), Academic Press, New York, 1959. 

E. X. Vrouwe, R. Luttge, W. Olthuis and A. van den Berg, Microchip analysis of lithium in blood using moving boundary electrophoresis and zone electrophoresis, Electrophoresis, 26 (2005) 3032-3042. 

The particularly high resolving power of moderately concentrated gel media is to a large extent a consequence of molecular sieving acting as an additional separative factor. For example, blood serum can be separated into about 25 components in polyacrylamide gel, but only into 5 components on filter paper or by moving boundary electrophoresis. 

The development of electrophoretic techniques afforded possibilities for fractionations based on charge density differences. Duxbury (1989) has reviewed applications of different electrophoretic separation methods, including zone electrophoresis, moving boundary electrophoresis, isotachophoresis, and isoelectric focusing (IEF). Preparative column electrophoresis (Clapp, 1957) and continuous flow paper electrophoresis (Hayes, 1960 summarized by Hayes et al., 1985) methods have been used to separate components isolated from sapric histosol soils. These techniques allowed separation of polysaccharides from the colored components the electrophoretograms of the colored components were diffuse, showing a continuum of components of different charge densities. 

Figure 4.15 Moving boundary electrophoresis pattern of normal human serum (Reproduced with permission from Bezkorovainy A. Basic Protein Chemistry. Springfield, IL Thomas, p. 20, 1970.)...

The term microelectrophoresis, or even better, microscopic electrophoresis, refers to a technique in which the motion of individual particles is followed, usually ultramicroscoplcally. Other terms refer to alternative ways of measurement, such as moving boundary electrophoresis, paper electrophoresis, laser-Doppler electrophoresis, gel electrophoresis, capillary electrophoresis, etc. see secs. 4.5 and 10. 

Regarding the determination of particle velocities, two approaches are possible. In the first, known as microelectrophoresis. Individual particles are made visible and their translation is monitored. This is the most basic procedure because the variation of the velocities over the particles can be measured. When microelectrophoresis is not feasible, the collective displacement of a large number of particles can be studied. This technique is known as moving boundary electrophoresis. We shall now discuss these modes of operation. 

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