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Electrophoresis

Electrophoresis is the process of directed movement of particles dispersed in a liquid in a constant electric field. Particles of the same substance carry charges of the same sign. In an electric field, positively charged particles move to the negative electrode - the cathode, negatively charged particles to the positive electrode - the anode. The movement of particles to the cathode is sometimes called cataphoresis, to the anode - anaphoresis. The speed of movement depends on the mass of the particles and their charge under the given conditions, due to which electrophoresis makes it possible to separate mixtures of substances into their constituent components.

electrophoresis scheme
Fig. 1. Scheme of free (frontal) electrophoresis. The position of the boundaries of the section: a - to experience; b - after the experience. 1 - electrodes; 2 - solvent ; 3 - protein solution.

There are the following types of electrophoresis. 1. Free (frontal) electrophoresis. In this case, electrophoresis is carried out in devices, an essential part of which is the U-shaped tube (Fig. 1). The bottom of the tube is filled with the test object, for example, a protein solution onto which the solvent is layered. Electrodes connected to a DC source are immersed in the solvent. In this case, electrically charged protein particles move to one of the electrodes, as a result of which the interface between the solution and the solvent rises in one knee (ascending boundary), and in the other it descends (descending boundary). Devices for free electrophoresis, equipped with a device for automatic registration of the movement of each component in the object under study, are used in the analysis of dispersed systems, the separation of individual components from them, as well as in the clinical study of blood serum .

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2. Electrophoresis on carriers (zone electrophoresis). Paper, gels of starch, agar, polyurethanes, etc. are used as carriers. In clinical laboratories, electrophoresis on paper has been particularly widely used for the study of blood serum: on a strip of special paper, impregnated with an appropriate buffer solution put a drop of serum. The ends of the strips are dipped in cups filled with this buffer solution and fitted with electrodes. When passing a direct current, individual serum proteins move along a strip at different speeds, and sometimes in different directions. After a certain time, the passage of current is stopped, the strip of paper is dried and treated with a reagent for protein. At the same time on paper electrophoregram revealed stained spots. By the number of spots, the amount of protein fractions is judged, and by the intensity of stain stains - the quantitative content of each protein fraction in the serum under study.

Recently, electrophoresis in thin layers of gels deposited on glass plates (disk electrophoresis) and placed in glass tubes has been widely used in research and in clinical diagnostics.

Electrophoretic studies . In clinical practice, zonal electrophoresis is used to study the protein composition of body fluids. Electrophoresis on paper is most often used as the simplest technique for performing. Electrophoresis in agar and starch gels is used in medical practice primarily in scientific research.

Using electrophoresis on paper, fractions of proteins, lipoproteins, glucoproteins, as well as protein fractions of urine, gastric juice, exudates, etc., are separated in blood. 2 (α 2 ) -, beta (β) - and gamma (γ) -globulins. Normally, their ratio is more or less constant. In some diseases, these ratios change, which may have diagnostic and prognostic value. For example, in acute inflammatory processes, the content of α 2 -globulins in the blood increases; in the period of development of immunity, the content of γ-globulins increases; in liver lesions, albumin content, etc., is reduced. In some diseases (for example, myeloma), pathological proteins (paraproteins) appear in the blood plasma, which can be detected using electrophoresis methods, which is of great diagnostic value.

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Electrophoresis is the phenomenon of the directional movement of ultramicroscopic and microscopic particles under the influence of an externally applied potential difference observed in suspensions, emulsions, colloidal solutions, solutions of high molecular compounds (for example, nucleic acid proteins, polysaccharides, etc.).

Electrophoresis is explained by the presence of microscopic and ultramicroscopic particles of electric charges, which arise as a result of selective adsorption by particles of ions from the surrounding dispersion medium or as a result of dissociation of ionogenic groups that make up the surface of the particles. The sign of the electric charge of a particle depends both on the nature of the particles themselves and on the composition of the dispersion medium. Particles of the same substance can be charged both positively and negatively when changing the composition of the dispersion medium. For example, protein macromolecules in solutions whose pH is less than the isoelectric point of this protein (see Ampholytes) are positively charged and move in an electric field to the negative pole - cathode, and in solutions whose pH is greater than the protein isoelectric point, its macromolecules are negatively charged and move to the positive pole - the anode. The movement of particles to the cathode is sometimes called cataphoresis, to the anode - anaphoresis.

Several methods are used to study electrophoresis.

The most accurate frontal electrophoresis, or the method of the moving boundary. The devices used for frontal electrophoresis are varied in design, but each of them includes an electrophoretic cuvette - usually a U-shaped tube (Fig. 1). The lower part of the tube is filled with the test liquid, for example, a colloidal solution, on which a dilute electrolyte solution with an electrical conductivity equal to that of the colloidal solution is layered. Non-polarizable electrodes connected to a constant current source are placed in open tube bends. When current is switched on, colloidal particles with a single front (as the particles in this colloidal solution have the same electric charge) move to one of the electrodes, resulting in an interface between the colloidal solution and the electrolyte solution in one bend of the tube (the ascending boundary), and in the other knee respectively falls (descending border). Moving the interface is easy to observe if the colloidal solution is colored. If the colloidal solution is colorless, the interface between it and the electrolyte solution can be made visible by illuminating the device from the side; however, the colloidal solution will be opalescent. Sometimes for this purpose they use the ability of colloidal solutions or solutions of high-molecular compounds to fluoresce under the action of ultraviolet rays.


Fig. 1. Electrophoretic cuvette (scheme): 1 - the test liquid; 2 - diluted electrolyte solution; 3 - electrodes.

The velocity U of the electrophoretic movement is related to the electrokinetic potential of the particles (the potential difference between the sliding boundary of the particle and the dispersion medium) by the relation:

where D is the dielectric constant of the dispersion medium, H is the potential drop of the electric field per unit length, n is 3.14, η is the viscosity of the dispersion medium and k is a constant depending on the shape of the particles (for small spherical particles, k — C, for cylindrical particles k — 4). The value of U / H, i.e. the speed of electrophoretic movement of particles, calculated for a potential drop of 1 V / cm, is called electrophoretic mobility. This value, which has a dimension of cm 2 in -1 sec -1, for proteins (near the isoelectric point) is 0.4–0.8 · 10 −4 , for erythrocytes of various animals - 1.0—1.7 · 10 -4 etc.

A perfect frontal electrophoresis technique for complex mixtures of proteins and other biopolymers was developed in 1937 by A. Tiselius. The Tizelpus device and its various modifications, which make it possible to automatically register the movement of the interface of each component in the mixture using special optical devices and special diagrams — electrophoregrams (Fig. 2), are widely used to study normal and pathological serums, determine the composition of protein mixtures, determine protein purity.


Fig. 2. Electrophoregrams on Tizelius: 1 - normal human serum; 2 - blood plasma in multiple myeloma; 3 - serum with nephrosis.

Zone electrophoresis is carried out in the medium of some indifferent carrier, for example, in agar, starch gels, gelatins, as well as on special grades of filter paper. Electrophoresis on paper (paper electrophoresis) is especially widespread in the study and separation of proteins, nucleic acids, sterols, amino acids, fatty acids and other biologically active substances. Electrophoresis on paper is carried out at a potential drop of about 15–20 V / cm and a current strength of 3-5 mA. The test mixture (usually 0.01-0.04 ml of solution in buffer) is applied in the form of a line in the middle of a strip of paper, the ends of which are immersed in electrode solutions equipped with non-polarizable electrodes. At the end of the electrophoresis, the paper is dried, if necessary, treated with a special reagent that stains the individual components of the mixture, which form several bands in accordance with the number of components. The intensity of the color of the bands of the electrophoregram can be judged on the relative amount of each component in the mixture.

The microscopic method of electrophoresis consists in determining the speed of movement of particles visible in a microscope in an electric field - bacterial cells, erythrocytes, particles of suspensions and emulsions, etc. Special micro cuvettes equipped with electrodes are used for microelectrophoresis.

See also Immunoelectrophoresis.