Electrophoresis physical principles

In this chapter we discuss the important separation methods based on chromatography, electrophoresis and centrifugation. We consider first the physical principles underlying each method and then their application in preparative and analytical work. Since these are core experimental techniques in biochemistry and molecular biology, they are discussed in depth so that they can be used without having to consult additional sources information. 

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... 

Countercurrent electrophoresis can be nsed to split a mixtnre of mobile species into two fractions by the electrical analog of elntria-tion. In such countercurrent electrophoresis, sometimes termed an ion still, a flow of the suspending flnid is maintained parallel to the direction of the voltage gradient. Species which do not migrate fast enough in the applied electric field will be physically swept out of the apparatus. An apparatus based mainly on this principle bnt nsing also natural convection currents has been developed (Bier, Electrophoresis, vol. II, Academic, New York, 1967). 

The word electrokinetic implies the joint effects of motion and electrical phenomena. We are interested in the electrokinetic phenomena that originate the motion of a liqnid within a capillary tube and the migration of charged species within the liquid that surrounds them. In the first case, the electrokinetic phenomenon is called electroosmosis whereas the motion of charged species within the solution where they are dissolved is called electrophoresis. This section provides a brief illns-tration of the basic principles of these electrokinetic phenomena, based on text books on physical chemistry [7-9] and specialized articles and books [10-12] to which a reader interested to stndy in deep the mentioned theoretical aspects should refer to. 

The electrophoretic separation technique is based on the principle that, under the influence of an applied potential field, different species in solution will migrate at different velocities from one another. When an external electric field is applied to a solution of charged species, each ion moves toward the electrode of opposite charge. The velocities of the migrating species depend not only on the electric field, but also on the shapes of the species and their environmment. Historically, electrophoresis has been performed on a support medium such as a semisolid slab gel or in nongel support media such as paper or cellulose acetate. The support media provide the physical support and mechanical stability for the fluidic buffer system. Capillary electrophoresis (CE) has emerged as an alternative form of electrophoresis, where the capillary wall provides the mechanical stability for the carrier electrolyte. Capillary electrophoresis is the collective term which incorporates all of the electrophoretic modes that are performed within a capillary. 

In 1977 another expression rolls machine known as the Elephant emerged. This adopted a new ceramic extrusion method utilizing electrophoresis following the Elephant principle. It cannot be formally classified as an extruder, rather being a machine for shaping by means of precipitating slurry in an electric field (electrophoresis) , yet the unusual features of this special process do in fact fulfil the requirements of column extrusion. The process could also be termed the extrusion of slurry The physical phenomenon of electrophoresis has been known since the 19 century, when Reuss discovered that particles suspended in water... 

Recently, the theory of dielectrophoresis was applied to explain the microscopic physics of the movement of pigments in electrophoretic image displays and to prove the discrepancies between theory and measurement [9], Dielectrophoresis is induced by the interaction of the electric field and the induced dipole and is used to describe the behavior of polarizable particles in a locally nonuniform electric field. For example, the phenomenon of the delay time can be explained by the principle of dielectrophoresis. In electrophoresis, when the backplane voltage is switched, the particles on the electrode have to move instantaneously under a given electric field. However, the particles need a removal time which results in a delay time in the switching process. The time constant to obtain an induced dipole from a particle at rest is derived by Schwarz s formula [10] and used to compute the dielectrophoretic force at its steady-state value. The force and the velocity fields under a nonuniform electric field due to the presence of pigments also help to estimate realistic values for physical properties. 

The above discussion makes clear that colloidal particles and polyelectrolytes cannot be separated by size during free solution electrophoresis. As a result, many electrophoretic size separations for chemistry and biology are performed in gels. The precision afforded by microfabrication has led to miniaturized version of these classic protocols, as well as a number of novel separation techniques that differ distinctly from the separation principles prevailing in gels [5]. In order to best understand the current research in microfluidic separations of colloids and poly electrolytes, in particular the important apphcations to protein and DNA separations, it is important to first understand the physics of gel electrophoresis. 

A comment should be made on the title of the book. Physical Chemistry of Macromolecules Basic Principles and Issues. The word basic refers to fundamental, meaning relatively timeless. In the selection of experimental methods and theories for each topic, the guideline was to include only those materials that do not change rapidly over time, for example. Pick s first law and second law in diffusion, Patterson s synthesis and direct method in x-ray crystallography, or those materials, though current, that are well estabhshed and frequently cited in the literature, such as the scaling concept of polymer and DNA sequencing by electrophoresis. The book is, therefore, meant to be a course of study. ... 

Electrophoresis is described in a number of useful works. Introduction to Paper Electrophoresis and Related Methods by M. Lederer (Elsevier, 1955) surveys its use in organic, inorganic and biological chemistry. Principles of Electrophoresis by R. Audubert and S. de Mende (Hutchinson, 1959) covers the physical chemistry of electrophoresis with detailed descriptions of apparatus and techniques. It is a useful introduction to new developments. 

The physical significance of the zeta potential is discussed in the following section. The suspension could be characterised by particle charge density, which can in principle be determined from the electrophoretic mobility, but which requires certain assumptions regarding the particle size and shape distribution and conductivity effects. The zeta potential is the most commonly used parameter for characterising a suspension, and can be determined from measurements of particle velocity or mobility in an applied field using commercially available electrophoresis cells. In practice electrophoretic mobilities are not easy to measure accurately, and since the Smoluchowski equation is based on a model of doubtful validity, the view sometimes expressed that "zeta potentials are difficult to measure and impossible to interpret" has a ring of truth but is probably unduly pessimistic. The Smoluchowski relation is valid provided that the double... 

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