Electrical separations electrophoresis

The possible formation of a dipole is a feature of covalent bonding but it is obvious that an ionic bond results in a definite unequal distribution of electrons within a molecule and such molecules (or ions) are extremely polar. However, the fact that they carry a definite charge enables additional separation techniques to be applied. The rate of migration in an electric field (electrophoresis) and the affinity for ions of opposite charge (ion-exchange chromatography) are extremely valuable techniques in the separation of ionic species. 

Because of their structural differences, isozymes may often be distinguished by separation in an electric field (electrophoresis) or by reactivity with selective antibodies. 

The acid-base properties, and hence ionic character, of peptides and proteins also can be used to achieve separations. Ion-exchange chromatography, similar to that described for amino acids (Section 25-4C), is an important separation method. Another method based on acid-base character and molecular size depends on differential rates of migration of the ionized forms of a protein in an electric field (electrophoresis). Proteins, like amino acids, have isoelectric points, which are the pH values at which the molecules have no net charge. At all other pH values there will be some degree of net ionic charge. Because different proteins have different ionic properties, they frequently can be separated by electrophoresis in buffered solutions. Another method, which is used for the separation and purification of enzymes, is affinity chromatography, which was described briefly in Section 9-2B. 

On the other hand, the intervening media used in electrophoresis have much lower conductivity, and an equivalent circuit for an electrophoresis cell includes a resistor between the capacitors at the electrode-solution interfaces. Across the support medium, potential is now (usually) a linear function of distance, and the electric field thus generated is responsible for driving the electrophoretic separation. Electrophoresis occurs at the electrodes used in electrophoresis, to maintain the... 

In electrophoresis, the net surface charge and the size of molecules separate them from one another in an electric field. Electrophoresis is one of the most effective methods of protein separation and purification. It has a very high resolving power to clearly separate charged protein molecules. This method of... 

It has been known for years that ions can be separated by differences in their rates of movement in an applied electric field (electrophoresis). Until fairly recently, electrophoresis was considered to be rather slow, a technique best reserved for separation of large organic ions and molecules. The realization that electrophoresis can be performed in a fused silica capillary has resulted in some dramatic changes. Now inorganic ions and small organic ions can be separated very quickly by this form of electrophoresis. Capillary zone electrophoresis (CZE), capillary electrophoresis (CE) and capillary ion electrophoresis (CIE) are the names most used to describe this type of separation. 

Figure 11. Separation of treated and untreated human erythrocytes by low-electric-field electrophoresis. Human erythrocytes (5 x 107) treated with neuraminidase are mixed with 5 x 107 untreated erythrocytes to a final volume of 5 ml and layered onto a linear gradient of 1.5-cm height. Electrophoresis proceeds for 25 min at 5°C at a constant current of 90 mA. An identical suspension is subjected to velocity sedimentation at unit gravity only. (O) Separation at unit gravity ( ) separation at unit gravity and by electrophoresis. Areas under the migration profile are depicted in shading. Gravity and electric forces act in the same direction (to the right). (Figures 8—11 reproduced with kind permission of the editor of Anal. Biochem.)...

Nitrocellulose membrane filters, nitrated papers used as membranes in protein engineering and the electrophoresis (electrical separation of particles in a liquid) of DNA and polymeric samples. 

Chemically, molecular conformations with large electric moments increase in concentration at the expense of those configurations with smaller moments. Secondly, the presence of electric fields increases the dissociation of weak acids and bases and promotes the separation of ion pairs into the corresponding free ions (dissociation field effect, second Wien effect). The free ions or ionized structures then may move in the direction of the electric field (electrophoresis) and a field-dependent stationary state in the ion distribution may be established. 

There are several different ways in which DNA can be detected and sized. The majority of which involve separation of different sized DNA fragments under the influence of an electric current — electrophoresis. The medium for gel electrophoresis varies from simple agarose to polyacrilamide. 

The determination of electrophoretic velocities may be carried out experimentally by the use of methods suitable for transport number measurements. Moving boundary techniques have proved useful despite the problem of a difficulty in selecting suitable indicator ions. Reliable estimates of electrophoretic velocities make possible the determination of zeta-potentials. Since colloids migrate at characteristic rates under the influence of an electric field, electrophoresis provides an important means of separation. Coatings, such as rubber or graphite, may be deposited on metal electrodes by this means and additives to these may be co-deposited. 

Electrophoresis is used primarily to analyze mix tures of peptides and proteins rather than individual ammo acids but analogous principles apply Because they incorporate different numbers of ammo acids and because their side chains are different two pep tides will have slightly different acid-base properties and slightly different net charges at a particular pH Thus their mobilities m an electric field will be differ ent and electrophoresis can be used to separate them The medium used to separate peptides and proteins is typically a polyacrylamide gel leading to the term gel electrophoresis for this technique... 

Capillary Electrophoresis. Capillary electrophoresis (ce) is an analytical technique that can achieve rapid high resolution separation of water-soluble components present in small sample volumes. The separations are generally based on the principle of electrically driven ions in solution. Selectivity can be varied by the alteration of pH, ionic strength, electrolyte composition, or by incorporation of additives. Typical examples of additives include organic solvents, surfactants (qv), and complexation agents (see Chelating agents). 

The physical separation of charge represented allows externally apphed electric field forces to act on the solution in the diffuse layer. There are two phenomena associated with the electric double layer that are relevant electrophoresis when a particle is moved by an electric field relative to the bulk and electroosmosis, sometimes called electroendosmosis, when bulk fluid migrates with respect to an immobilized charged surface. 

The heating effect is the limiting factor for all electrophoretic separations. When heat is dissipated rapidly, as in capillary electrophoresis, rapid, high resolution separations are possible. For electrophoretic separations the higher the separating driving force, ie, the electric field strength, the better the resolution. This means that if a way to separate faster can be found, it should also be a more effective separation. This is the opposite of most other separation techniques. 

Differences in mobilities of ions, molecules, or particles in an electric field can be exploited to perform useful separations. Primary emphasis is placed on electrophoresis and dielec trophoresis. Analogous separation processes involving magnetic and centrifugal force fields are widely apphed in the process industiy (see Secs. 18 and 19). 

Modes of Operation There is a close analogy between sedimentation of particles or macromolecules in a gravitational field and their elec trophoretic movement in an electric field. Both types of separation have proved valuable not only for analysis of colloids but also for preparative work, at least in the laboratoiy. Electrophoresis is applicable also for separating mixtures of simple cations or anions in certain cases in which other separating methods are ineffectual. 

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. 

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