electrophoresis

1. The migration of charged colloidal particles or molecules through a solution under the influence of an applied electric field usually provided by immersed electrodes. Also called cataphoresis.
2. A method of separating substances, especially proteins, and analyzing molecular structure based on the rate of movement of each component in a colloidal suspension while under the influence of an electric field.

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The migration of electrically charged particles in solution or suspension in the presence of an applied electric field. Each particle moves toward the electrode of opposite electrical polarity. For a given set of solution conditions, the velocity with which a particle moves divided by the magnitude of the electric field is a characteristic number called the electrophoretic mobility. The electrophoretic mobility is directly proportional to the magnitude of the charge on the particle, and is inversely proportional to the size of the particle. An electrophoresis experiment may be either analytical, in which case the objective is to measure the magnitude of the electrophoretic mobility, or preparative, in which case the objective is to separate various species which differ in their electrophoretic mobilities under the experimental solution conditions.

Gel techniques

Electrophoresis was first employed as an experimental technique by Arne Tiselius in 1937. The apparatus of Tiselius detected electrophoretic motion by the moving-boundary method, in which a boundary is created between the solution of particles to be examined and a sample of pure solvent. As the particles migrate in an electric field, the boundary between solution and solvent can be observed to move, and if there are a number of species in the solution with different electrophoretic mobilities, a series of boundaries of various shapes and magnitudes can be detected. The moving-boundary method was used for three decades to separate complex mixtures of charged macromolecules in solution and to study the physical characteristics of solutions of proteins and other macromolecules of biological and industrial importance.

The resolving power of electrophoresis was greatly improved by the introduction of the use of gel supporting media. The gel matrix prevents thermal convection caused by the heat which results from the passage of electric current through the sample. The absence of convection reduces greatly the mixing of the various parts of the sample, and therefore allows for more stable separation. The dimensions of the cross-links of the gel may also provide a molecular sieving effect, which increases the resolving power of the electrophoretic separation of molecules of different size. In addition, the gel media may support a gradient of a separate reagent, which assists in the separation of macromolecules. Gradients of pH and of reagents of various types may be combined in two-dimensional arrays for even greater resolving power. A very successful derivative of the gel technique is the determination of the molecular weights of protein molecules by electrophoresis of the molecules in a gel medium which contains substantial amounts of detergent. The detergent denatures the protein molecules, changing them from globular, compact structures to long, flexible polymers which are coated with detergent molecules. These polymers move in the electric field through the gel medium with a velocity which is determined by the length of the polymer, and therefore by the molecular weight of the protein unit. This method is the most common technique for the determination of molecular weights of proteins in biochemical studies. See also Gel; pH; Protein.

Isoelectric focusing

An important variation of the electrophoresis technique is isoelectric focusing. In this technique the medium supports a pH gradient which includes the isoelectric pH of the species being studied. Many charged macromolecules have both positive and negative charges on their surfaces, and the electrophoretic mobility is related to the net excess of charge of one type or the other. As the pH becomes more acidic, the number of positive charges increases, and as the pH becomes more basic, the number of negative charges increases. For each molecule of this type, there is one pH at which the net charge on the surface is zero, so that the molecule does not move when an electric field is applied and thus has an electrophoretic mobility of zero. This pH is called the isoelectric pH. If the molecule is introduced into a pH gradient which includes its isoelectric pH, it will migrate to the position of the isoelectric pH and then become stationary. In this way, all molecules of a given isoelectric pH will migrate to the same region—hence the term isoelectric focusing. The method of isoelectric focusing is particularly good for the analysis of microheterogeneity of protein species and other species which may differ slightly in their chemical content. See also Isoelectric point.

Laser applications

Application of the optical laser to electrophoretic detection resulted in the development of a technique which can be used for analytical electrophoresis experiments on particles of all sizes. The basic principle is that the highly monochromatic (single-frequency) laser light impinges upon the particles and is scattered from the particles in all directions. When observing the laser light which has been scattered from a moving particle, one can detect that there is a slight shift in the frequency of the light as a result of the motion of the particle. The application of the laser Doppler principle to electrophoresis experiments, often called electrophoretic light scattering (ELS), is an important method for the rapid determination of electrophoretic velocities. Electrophoretic light scattering has been used for the study of many types of living cells, cell organelles, viruses, proteins, nucleic acids, and synthetic polymers. See also Electrolytic conductance.

Capillary electrophoresis

Electrophoresis can be performed in a capillary format. A typical system consists of two reservoirs and a capillary filled with a buffer solution. A high voltage is applied across the capillary by using a high-voltage power supply. The very small diameter capillaries (typically 5–100 micrometers) employed in this technique allow for efficient heat dissipation. Therefore, much higher voltages can be employed than those used in slab gel electrophoresis, leading to faster, more efficient separations. Compounds are separated on the basis of their net electrophoretic mobilities.

Most often, the detector is placed on line and analytes are detected as they flow past the detector. Spectroscopic detection (ultraviolet and laser-based fluorescence) is usually performed in this manner by using the capillary itself as the optical cell. Alternatively, detectors can be placed off line (after the column). In this case, the detector is isolated from the applied electric field through the use of a grounding joint. Electrochemical detection and mass spectroscopic detection are generally accomplished in this manner, since the electric field can interfere with the performance of these detectors.

Capillary zone electrophoresis is the simplest and most widely used form of capillary electrophoresis. The capillary is filled with a homogeneous buffer, and compounds are separated on the basis of their relative charge and size. Most often, fused silica capillaries are employed. In this case, an electrical double layer is produced at the capillary surface due to the attraction of positively charged cations in the buffer to the ionized silanol groups on the capillary wall. In the presence of an electric field, the cations in the diffuse portion of this double layer move toward the cathode and drag the solvent with them, producing an electroosmotic flow. The resulting flow profile is flat rather than the parabolic shape characteristic of liquid chromatography. This flat flow profile causes analytes to migrate in very narrow bands and leads to highly efficient separations. The electroosmotic flow is also pH dependent, and it is highest at alkaline pH values.

In most cases, the electroosmotic flow is the strongest driving force in the separation, and all analytes, regardless of charge, migrate toward the cathode. Therefore, it is possible to separate and detect positive, negative, and neutral molecules in the same electrophoretic run, if the detector is placed at the cathodic end. Negatively charged compounds are attracted to the anode but are swept up by the electroosmotic flow and elute last. Neutral molecules, which are not separated from each other in capillary zone electrophoresis, elute as a single band with the same velocity as the electroosmotic flow. Positive compounds have positive electrophoretic mobilities in the same direction as the electroosmotic flow, and they elute first (see illustration). Capillary zone electrophoresis is generally employed for the separation of small molecules, including amino acids, peptides, and small ions, and for the separation of drugs, their metabolites, and degradation products.

Capillary zone electrophoresis. (a) Separation mechanism showing electrophoretic mobility of the positive ion (μM⁺) and negative ion (μM⁻); N is a neutral molecule. (b) Migration order of the ions.

All capillary electrophoresis methods have the advantage of the ability to analyze small volumes (typical injection volumes are 1–50 nanoliters). This makes it possible to analyze very small samples or to use the same sample for several different analyses. One unique application of this technique is the determination of amino acids and neutrotransmitters in single cells.

Alternating-field electrophoresis

Alternating-field agarose gel electrophoresis is a technique for separating very large molecules of deoxyribonucleic acid (DNA); fragments of DNA ranging in size from 30 to 10,000 kilobasepairs (kb) can be resolved. For the molecular biologist, this is a considerable advance over conventional agarose gel electrophoresis, which is limited to the resolution of less than 50 kb.

Conventional gel electrophoresis employs a single pair of electrodes to generate an electric field that is constant in both time and direction and that is uniform across the gel. DNA molecules are negatively charged with a uniform charge-to-mass ratio and thus migrate steadily toward the positive electrode. Although DNA is a linear molecule, in solution it tends to collapse into a random coil configuration. Agarose is a porous material that acts like a sieve, retarding the movement of the DNA; the larger the molecule, the more the retardation, and thus the molecules separate on the basis of size. However, above approximately 50 kb, the dimensions of the random coil are larger than the pore size of the agarose. The DNA can no longer be sieved through the gel, and resolution is lost.

Contrary to conventional electrophoresis, alternating-field electrophoresis does not use a constant electric field but one which regularly alternates in direction. With each change in field direction, the DNA molecules attempt to reorient themselves. When this happens, an end or a small loop of the molecule, which has dimensions much smaller than those of the random coil of the entire molecule, may find itself positioned by a pore in the agarose. The electric field can then pull the DNA by the end through the hole. When the field regularly alternates from one direction to another, the DNA regularly reorients and is pulled through an adjacent hole.

Not all molecules in the system will make equal progress under these conditions, because not all molecules can reorient themselves with equal speed. The larger the molecule, the more time it requires in a given field strength (determined by the applied voltage) to change directions and the less time it has left to move.

The movement of charged suspended particles through a liquid medium in response to changes in an electric field.

Electrophoresis is a technique used to separate biological molecules, such as nucleic acids, carbohydrates, and amino acids, based on their movement due to the influence of a direct electric current in a buffered solution. Positively charged molecules move toward the negative electrode, while negatively charged molecules move toward the positive electrode.

Diseases caused by microorganisms are a threat to national security. Even in countries with well-developed healthcare systems, a massive outbreak can strain healthcare infrastructure. In other countries that are less wealthy and more politically volatile, the ravages of disease can sow the seeds of resentment against the more wealthy countries of the West. Thus, it is in a country's best interests to combat infectious diseases. One strategy is to examine the relevant microorganisms, particularly to find out the component(s) that are responsible for the infection. For many microbes, proteins are an important factor in the development of a disease. Proteins can function as receptors, to allow the microorganism to adhere to the surface of a host cell. As well, the toxins produced by microbes such as Escherichia coli O157:H7 and Vibrio chlorerae are proteins. Methods that can "dissect" microorganisms into their components, and which can compare a non-disease causing strain of a microbe to a diseasecausing strain to see what they differences are, is a valuable approach to fighting infectious disease. Electrophoresis is especially well suited to this role. Furthermore, specialized types of electrophoresis (i.e., pulsed field electrophoresis) allow the genetic material of the microorganism to be examined. Thus, electrophoresis can reveal much detail at the molecular level.

Electrophoresis is a sensitive analytical form of chromatography. Under the influence of an electrical field charged molecules can be separated from one another as they pass through a gel. The degree of separation and rate of molecular migration of mixtures of molecules depends upon a variety of factors, which can be tailored depending upon the intent of the separation. For example, conditions can be established that allow molecules of very large mass, but which differ from each other by only a fraction, to be visually separated. The factors that influence molecular separation include the individual size and shape of the molecules, their molecular charge, strength of the electric field, the type of support medium used (e.g., gels made of cellulose acetate, starch, paper, agarose, polyacrylamide) and the conditions of the medium (e.g., ion strength and concentration, pH, viscosity, temperature).

The advent of electrophoresis revolutionized the methods of protein analysis. Swedish biochemist Arne Tiselius was awarded the 1948 Nobel Prize in chemistry for his pioneering research in electrophoretic analysis. Tiselius studied the separation of serum proteins in a tube (subsequently named a Tiselius tube) that contained a solution subjected to an electric field.

In electrophoresis, the electric charge often is passed through what is known as a support medium. As summarized above, various support media can be used. They all share the trait that they are a three-dimensional arrangement of intertwined strands, which produces holes (or pores) through the gel matrix. Such matrices act as a physical sieve for macromolecules.

In general, the medium is mixed with a chemical mixture called a buffer. The buffer carries the electric charge that is applied to the system. The medium/buffer matrix is placed in a tray. Samples of molecules to be separated are loaded into wells or slots that have been formed at one end of the matrix. As electrical current is applied to the tray, the matrix takes on this charge and develops positively and negatively charged ends. As a result, molecules that are negatively charged such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein are pulled toward the positive end of the gel.

Because molecules have differing shapes, sizes and charges they are pulled through the matrix at different rates and this, in turn, causes a separation of the molecules. Generally, the smaller and more charged a molecule, the faster the molecule moves through the matrix.

Intact DNA is so large that it cannot move through the pores of a gel (although the technique of pulsed field electrophoresis does allow very large pieces of DNA to be examined). When DNA is subjected to electrophoresis, the DNA is first cut into smaller pieces by restriction enzymes. Restriction enzymes recognize specific sequences of the building blocks of the DNA and cut the DNA at the particular site. There are many types of restriction enzymes, and so DNA can be cut into many different patterns. After electrophoresis, the pieces of DNA appear as bands (composed of similar length DNA molecules) in the electrophoresis matrix.

Proteins have net charges determined by charged groups of the amino acids from which they are constructed. Proteins can also be amphoteric compounds (a compound that can take on a negative or positive charge depending on the surrounding conditions.) A protein in one solution might carry a positive charge in a particular medium and thus migrate toward the negative end of the matrix. In another solution the same protein might carry a negative charge and migrate toward the positive end of the matrix. For each protein there is a pH in which the protein molecule has no net charge (the isoelectric point). By varying the pH in the matrix, additional refinements in separation are possible.

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis techniques pioneered in the 1960s provided a powerful means of protein separation. Still, because proteins of similar mass did not always clearly separate into discrete bands in the gel only small numbers of molecules could be separated.

The development in the 1970s of a two-dimensional electrophoresis technique allowed greater numbers of molecules to be separated. Two-dimensional electrophoresis is actually the fusion of two separate separation procedures. The first separation (dimension) is achieved by isoelectric focusing (IEF) that separates protein polypeptide chains according to the arrangement of amino acids that comprise a chain. IEF is based on the fact that proteins will, when subjected to a pH gradient, move to their isoelectric point. The second separation is achieved via SDS slab gel electrophoresis, which separates the molecule by molecular size. Instead of broad, overlapping bands, the result of this two-step process is the formation of a two-dimensional pattern of spots, each comprised of a unique protein or protein fragment. These spots are subsequently subjected to staining and further analysis.

Electrophoresis can be combined with the prior addition of a radioactive food source to the culture of bacteria. The bacteria will use the food to make new proteins, which will be radioactive. Following electrophoresis, the gel can be placed in contact with x-ray film. The radioactive bands or spots will register on the film, and so will determine what proteins were being made at the time of the experiment.

There are many other variations on gel electrophoresis with wide-ranging applications. These specialized techniques include Southern, Northern, and Western Blotting. Blots are named according to the molecule under study. In Southern blots, DNA is cut with restriction enzymes then probed with radioactive DNA. In Northern blotting, RNA is probed with radioactive DNA or RNA. Western blots target proteins with radioactive or enzymatically-tagged antibodies.

Modern electrophoresis techniques now allow the identification of DNA sequences that are the same, and have become an integral part of research into gene structure, gene expression, and the diagnosis of heritable diseases. Electrophoretic analysis also allows the identification of bacterial and viral strains and is finding increasing acceptance as a powerful forensic tool.

Further Reading

Books

Birren, Bruce W., and Eric Hon Cheong Lai. Pulsed Field Electrophoresis: A Practical Guide. San Diego: Academic Press, 1997.

Rabilloud, Thierry. Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods (Principles and Practice). Berlin: Springer Verlag, 2000.

Westermeier, Reiner. Electrophoresis in Practice. Weinheim: Vch Verlagsgesellschaft 2001.

Electronic

Colorado State University. "Gel Electrophoresis of DNA and RNA." Biomedical Hypertextbooks. January 15,2000. <http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/>(5 January 2003).

The movement of charged particles suspended in a liquid through various media, e.g. paper, cellulose acetate, gel, liquid, under the influence of an applied electric field.
The various charged particles of a particular substance migrate in a definite and characteristic direction—toward either the anode or the cathode—and at a characteristic speed. This principle has been widely used in the separation of proteins and nucleic acids and is therefore valuable in the study of diseases in which the serum and plasma proteins are altered. See also immunoelectrophoresis.

• SDS–polyacrylamide gel e. (SDS–PAGE) — a procedure that revolutionized the analysis of complex mixtures of proteins. The proteins are solubilized by the powerful, negatively charged detergent sodium dodecyl sulfate (SDS) which causes proteins to unfold into extended, single polypeptide chains. A reducing agent such as mercaptoethanol is usually added to break disulfide bonds. The constituent polypeptides are then electrophoresed through an inert matrix of highly cross-linked gel of polyacrylamide. The pore size of the gel can be varied by altering the concentration of polyacrylamide.
• two-dimensional gel e. — a SDS–polyacrylamide gel electrophoresis run, first in one direction, then again at right angles. In the first dimension an isoelectric-focusing gel is run and in the second dimension the proteins are separated in SDS–PAGE. A greater number of individually different proteins can be resolved in a highly repeatable fingerprint-like pattern.

For specific types of electrophoresis (for example, the process of administering medicine, iontophoresis), see Electrophoresis (disambiguation).

Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.^{[1][2][3][4][5][6]} This electrokinetic phenomenon was observed for the first time in 1807 by Reuss,^[7] who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid.

Illustration of electrophoresis

Illustration of electrophoresis retardation

Contents 1 Theory 2 See also 3 References 4 External links 5 Further reading

Theory

The dispersed particles have an electric surface charge, on which an external electric field exerts an electrostatic Coulomb force. According to the double layer theory, all surface charges in fluids are screened by a diffuse layer of ions, which has the same absolute charge but opposite sign with respect to that of the surface charge. The electric field also exerts a force on the ions in the diffuse layer which has direction opposite to that acting on the surface charge. This latter force is not actually applied to the particle, but to the ions in the diffuse layer located at some distance from the particle surface, and part of it is transferred all the way to the particle surface through viscous stress. This part of the force is also called electrophoretic retardation force.

Considering the hydrodynamic friction on the moving particles due to the viscosity of the dispersant, in the case of low Reynolds number and moderate electric field strength E, the speed of a dispersed particle v is simply proportional to the applied field, which leaves the electrophoretic mobility μ_e defined as:

The most known and widely used theory of electrophoresis was developed in 1903 by Smoluchowski^[8]

,

where ε_r is the dielectric constant of the dispersion medium, ε₀ is the permittivity of free space (C² N⁻¹ m⁻²), η is dynamic viscosity of the dispersion medium (Pa s), and ζ is zeta potential (i.e., the electrokinetic potential of the slipping plane in the double layer).

The Smoluchowski theory is very powerful because it works for dispersed particles of any shape at any concentration. Unfortunately, it has limitations on its validity. It follows, for instance, from the fact that it does not include Debye length κ⁻¹. However, Debye length must be important for electrophoresis, as follows immediately from the Figure on the right. Increasing thickness of the double layer (DL) leads to removing point of retardation force further from the particle surface. The thicker DL, the smaller retardation force must be.

Detailed theoretical analysis proved that the Smoluchowski theory is valid only for sufficiently thin DL, when particle radius a is much greater than Debye length:

.

This model of "thin Double Layer" offers tremendous simplifications not only for electrophoresis theory but for many other electrokinetic theories. This model is valid for most aqueous systems because the Debye length is only a few nanometers there. It breaks only for nano-colloids in solution with ionic strength close to water

The Smoluchowski theory also neglects contribution of surface conductivity. This is expressed in modern theory as condition of small Dukhin number

Du < < 1

In the effort of expanding the range of validity of electrophoretic theories, the opposite asymptotic case was considered, when Debye length is larger than particle radius:

κa < 1.

Under this condition of a "thick Double Layer", Huckel ^[9] predicted the following relation for electrophoretic mobility:

.

This model can be useful for some nanoparticles and non-polar fluids, where Debye length is much larger than in the usual cases.

There are several analytical theories that incorporate surface conductivity and eliminate the restriction of a small Dukhin number, pioneered by Overbeek^[10] and Booth^[11]. Modern, rigorous theories valid for any Zeta potential and often any κa stem mostly from Dukhin-Semenikhin theory^[12]. In the thin Double Layer limit, these theories confirm the numerical solution to the problem provided by O'Brien and White.^[13]

Recent molecular dynamics simulations nonetheless suggest that a surface charge is not always required for electrophoresis to occur, and that even neutral particles can migrate in an electric field due to the molecular structure of water at the interface.^[14].

See also

• Affinity electrophoresis
• Capillary electrophoresis
• Electroblotting
• Electrophoretic display
• Electrophoretic light scattering
• Electrophoretogram
• Gel electrophoresis
• Isotachophoresis
• Onsager reciprocal relations
• Protein electrophoresis
• SDS PAGE

References

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1. ^ Lyklema, J. “Fundamentals of Interface and Colloid Science”, vol.2, page.3.208, 1995
2. ^ Hunter, R.J. "Foundations of Colloid Science", Oxford University Press, 1989
3. ^ Dukhin, S.S. & Derjaguin, B.V. "Electrokinetic Phenomena", J.Willey and Sons, 1974
4. ^ Russel, W.B., Saville, D.A. and Schowalter, W.R. “Colloidal Dispersions”, Cambridge University Press,1989
5. ^ Kruyt, H.R. “Colloid Science”, Elsevier: Volume 1, Irreversible systems, (1952)
6. ^ Dukhin, A.S. and Goetz, P.J. "Ultrasound for characterizing colloids", Elsevier, 2002
7. ^ Reuss, F.F. Mem.Soc.Imperiale Naturalistes de Moscow, 2, 327 1809
8. ^ M. von Smoluchowski, Bull. Int. Acad. Sci. Cracovie, 184 (1903)
9. ^ Huckel, E., Physik.Z., 25, 204 (1924)
10. ^ Overbeek, J.Th.G., Koll.Bith, 287 (1943)
11. ^ Booth, F. Nature, 161, 83 (1948)
12. ^ Dukhin, S.S. and Semenikhin, N.M. Koll.Zhur., 32, 366 (1970)
13. ^ O'Brien, R.W. and White, L.R. J.Chem.Soc.Faraday Trans. 2, 74, 1607, (1978)
14. ^ Knecht et al., J. Col. Int. Sc. 318, p. 477, 2008

External links

• List of relative mobilities
• Handbook of Physics and Chemistry
• Dispersion Technology

Further reading

• http://gslc.genetics.utah.edu/units/activities/electrophoresis/
• Voet and Voet (1990) Biochemistry. John Whiley & sons.
• Jahn, G.C., Hall, D.W., and Zam, S.G. (1986) A comparison of the life cycles of two Amblyospora (Microspora: Amblyosporidae) in the mosquitoes Culex salinarius and Culex tarsalis Coquillett. J. Florida Anti-Mosquito Assoc. 57, 24–27.
• Khattak M.N. and Matthews R.C. (1993) Genetic relatedness of Bordetella species as determined by macrorestriction digests resolved by pulsed-field gel electrophoresis. Int. J. Syst. Bacteriol. 43(4), 659-64.
• Barz, D.P.J. and Ehrhard. P. (2005) Model and verification of electrokinetic flow and transport in a micro-electrophoresis device. Lab Chip 5, 949–958.
• Shim, J., Dutta, P., and Ivory, C.F. (2007) Modeling and simulation of IEF in 2-D microgeometries. Electrophoresis 28, 527–586.

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