Electrophoresis

Type of physical science: Chemistry

Field of study: Chemical methods

Electrophoresis is a process in which charged particles are transported through a fluid encompassed by an electric field. The technique may be used for purification of macromolecules, or in the study of properties such as size, charge, or shape.

Overview

Electrophoresis is defined as the migration of charged molecules under the influence of an electric field. Many types of molecules or particles, when placed in a polar (charged) solution, exhibit an electric charge. Consequently, when an electric current is run between two electrodes, the molecules in the solution will migrate to the appropriate pole.

Electrophoresis represents one of several related electrokinetic processes that illustrate the interaction between the charged surface of the dissolved material and counter-charges of the polar medium: electrophoresis, which follows the application of the electric field to the movement of dissolved molecules; its counterpart, electro-osmosis, the movement of liquid relative to a charged surface; and the opposite of electrophoresis, defined as sedimentation potential, which involves the actual creation of the electric field resulting from the movement of charged materials relative to a stationary medium. Of these phenomena, the one most readily applicable is the procedure of electrophoresis.

An early example of the principles involved was illustrated by Sir Oliver Joseph Lodge in 1886. His experiments measured the movement of the ions of an acid and base mixture, suspended in a gel subjected to an electric field. The motion of the charges could be followed with the addition of a pH indicator. This evolved into the procedure of moving boundary electrophoresis, later perfected by the Swedish scientist Arne Tiselius.

The major problem associated with the moving boundary procedure is that complete separation of individual components in the mixture is rarely achieved. A more analytical approach, later developed by Emmett Durrum in the United States, and Theodore Wieland and Fritz Turba in Germany, involved the migration of charged particles through a solid or gel supporting framework. This process resulted in the separation of molecules or particles into discrete zones, a procedure termed zone electrophoresis. Compared with the moving boundary method, zone electrophoresis also had the advantage of simplicity and the use of relatively inexpensive equipment.

Four requirements are necessary for the separation of macromolecules by electrophoresis. The first is the presence of charges on the molecules to undergo separation.

Many biological materials, particularly proteins and nucleic acids, are present as charged polymers. The relative charge on the molecule is dependent on the pH of the buffer in which the material is dissolved. As the pH of a solution is increased, becoming more alkaline, the proton concentration of the mixture is decreased. Hence, the molecules may exhibit a net negative charge. On the other hand, as the pH of the solution is decreased, becoming more acidic and resulting in an increase in proton concentration, the mass molecules may now exhibit a net positive charge. Therefore, depending on the particular pH, a protein may migrate to either pole.

If no net charge is present on the molecule, a condition referred to as the isoelectric point, the molecule may not migrate at all.

The second requirement necessary for electrophoresis is the presence of an electric field. If the suspended molecule is under the influence of an electric field, it will migrate at a constant velocity, and at a rate proportional to the applied voltage (the higher the voltage applied, the more rapid the electrophoretic separation). This proportional relationship of velocity to applied voltage is sometimes the source of confusion, inasmuch as it is often observed that velocity is proportional to current, a factor important in reproduction of experiments. (It should be noted that, according to Ohm's law, I = V/R, that is, current equals voltage divided by the resistance across the medium.) The explanation is that the velocity is not directly proportional to the voltage applied to the system, but rather to the voltage as perceived by the dissolved molecules. The effects of heat must also be taken into consideration. First, if either the current or voltage is too high, the temperature of the support medium may increase to a level that could alter its properties (the Joule heating effect, wattage = V x I). Increased heat could also lead to evaporation of the liquid buffer, resulting in an increase in resistance, and even possible denaturation of protein during electrophoresis. To minimize the problem, direct cooling of the apparatus is routinely carried out. The difficulties associated with heat, while minimized, can never be completely eliminated; hence, resistance changes with time, with the potential for subsequent alterations in velocity.

The third requirement of electrophoresis is a buffer system. Buffers are routinely used to maintain a constant pH and to carry the current. In addition, however, ions dissociated from a buffer may exert an effect on the electrophoretic mobility of a molecule, particularly that of a protein. For example, proteins may contain free acid (carboxyl) and basic (amide) groups, a property that renders them amphoteric. Ions associated with particular buffers may interact with these groups, forming salts, a process that is dependent on pH. The significance of this property is that alteration of electrophoretic mobilities associated with the proteins may occur.

The final requirement is the support medium, which can include cellulose acetate of paper strips, or various types of gel compounds. The application and properties associated with these materials in carrying out zone electrophoresis are discussed in more detail below.

Applications

Study of the chemical characteristics of macromolecules requires their isolation and purification. Some large molecules, particularly those, such as proteins or nucleic acids, that are intrinsic to the field of biochemistry, are often found in nature as charged polymers.

Consequently, a useful method for the study of such molecules is to follow their movement within the influence of an electric field: in other words, electrophoresis.

Microelectrophoresis involves the study of the distribution of electrical charges on the surface of microscopic particles. Generally, these particles are objects that can be seen with the use of a microscope, and include bacteria, protozoa, and blood cells. The properties of smaller molecules such as proteins have been observed by attaching these particles to tiny quartz spheres introduced into the apparatus. The apparatus has generally consisted of a thin observation cell, hooked to electrodes and placed within the optical field of a microscope. While the procedure has been useful in the past in the study of surface properties of particles, soluble proteins, and even enzymes, since the early 1960's, the technique has largely been surpassed by electrophoretic techniques involving either the moving-boundary (Tiselius) method, or zone electrophoresis.

Moving-boundary electrophoresis represents the oldest method of separation of macromolecules. Its usefulness as an analytical procedure lies in its ability to allow measurement of the mobility of charged particles through a fluid, as a function of time. During the procedure, a distinct boundary between the particles to be analyzed and the buffer against which they are layered is apparent. Following the passage of an electric current through the solution, the boundary will migrate at a velocity determined in part by the charge distribution on the dissolved material (solute). If the solute consists of a mixture of charged particles, a series of boundaries will become apparent.

The moving-boundary procedure of electrophoresis is usually carried out using a variation of the Tiselius cell, a U-shaped vessel with electrodes attached to the arms. The method has historically been used in determining the mobilities and isoelectric points of proteins such as those found in blood serum. For example, the serum from patients with certain hemoglobin anomalies may show gross deviation when compared with that from normal individuals. The procedure has also been used in testing the homogeneity of purified materials, and even in the fractionation of such materials.

The disadvantages associated with the moving-boundary method have largely resulted in the abandonment of this procedure in favor of that of zone electrophoresis. First, as an analytical tool, the procedure generally does not result in complete separation of molecules within a mixture. In addition, the usefulness of quantitative determination of mobilities is limited, and can be better supplied by other methods. Finally, the procedure requires relatively expensive apparatus to produce limited benefits. Most types of analysis, including clinical studies, can be carried out using the newer method of zone electrophoresis.

Zone electrophoresis has largely become the method of choice in the analysis of particles based on their charge distribution. Small samples can be evaluated, the equipment is simpler and less expensive, and, because only small quantities of materials are necessary, isolation is more easily achieved.

The term "zone electrophoresis" actually encompasses a number of variations in which the suspension of molecules is applied to a porous, semisolid material, followed by the application of an electric current. The molecules then migrate through the supporting medium.

Some materials, such as starch, agarose, or acrylamide, actually act as molecular sieves, aiding in the separation of the dissolved material. Because only a small quantity of sample is applied to the support medium (within a small "zone"), complete separation of solutes is readily achieved.

The support medium originally and most commonly used is cellulose (paper).

Generally, the procedure involves first wetting the paper to maintain a constant pH, then adding a small spot of the sample, usually a mixture of amino acids or short protein chains. The ends of the paper are placed in the presence of electrodes, and the electric current is applied to the strip.

When the separation is completed, the paper is removed and analyzed. If the sample is radioactive, minimal quantities of material are required, and fractionation becomes even more qualitative. Various other procedures exist for detection of sample materials. The use of low voltage (approximately 20 volts per centimeter of paper) is preferred for separation of small molecules such as amino acids, while high-voltage electrophoresis (200 volts per centimeter) is frequently employed for separation of larger polymers.

Because of the presence of hydroxyl (-OH) groups in cellulose, some biological molecules may absorb to paper, impeding resolution. For this reason, a cellulose acetate membrane is often substituted for paper. The transparency of cellulose acetate, and ease of elution of the sample following electrophoresis, render additional advantages to this medium over paper.

The simplicity and accuracy of celluose acetate strip electrophoresis resulted in its replacing the moving-boundary method for analysis of serum proteins such as the globulins, or albumin, and other small biological molecules. But this procedure too is subject to certain disadvantages. Both paper and celluose acetate become brittle when dry, and may be difficult to handle. In addition, superior resolution can be achieved by employing the technique of gel electrophoresis.

Since the 1970's, the use of gel materials such as starch, agarose, and acrylamide have constituted the most widely used electrophoretic systems. The most significant difference between zone electrophoresis on support media such as paper or cellulose acetate, and that on gels, is the molecular sieving action of gels, which adds a dimension to separation. For example, while separation of blood serum on cellulose acetate may demonstrate five to seven blood proteins, that serum sample may be resolved into more than twenty proteins by gel electrophoresis.

The original gel procedures employed the use of starch gels, prepared from potato starch, that had been heated in a buffer solution. The gel solution was poured into a mold or trough and was allowed to cool. The sample could then be applied, and electrophoresis carried out. Proteins, depending on their charge, could migrate to either electrode. The use of starch gels has been particularly helpful in the study of isozymes, enzymes with similar function but that differ slightly in charge because of small differences in amino acid sequences. The ease of elution without loss of enzyme activity has rendered starch superior to acrylamide as a support medium, under certain circumstances.

For general separation of proteins or nucleic acids, the use of agarose or acrylamide has replaced that of starch. Polyacrylamide, prepared by cross-linkage of acrylamide with bisacrylamide, is the most commonly used support medium. Its molecular sieving action for small nucleic acids or proteins is easily regulated by varying the concentration of gel material.

For separation of larger nucleic acids, the acrylamide can be replaced by either agarose or an acrylamide-agarose mixture. For these reasons, polyacrylamide gel electrophoresis has become the procedure of choice for analysis of proteins or small nucleic acids.

Since most DNA (deoxyribonucleic acid) molecules are too large to penetrate the molecular sieve associated with acrylamide, the use of agarose as a separation medium was introduced in the 1970's. Agarose has proved effective both as a support medium and as a sieve that can resolve molecules that differ as little as 1 percent in their molecular weight. Elution or electrophoretic transfer of DNA from the gel to membrane filters (for example, nitrocellulose) has also allowed the development of increasingly technical molecular analysis, including DNA sequencing. Additional variations of electrophoresis have also resulted in procedures for large-scale preparative isolations, possessing the advantages of working with large quantities of material, with the ability to resolve individual components of those mixtures. In effect, electrophoresis has come full circle.

Context

The understanding of the principles behind electrophoresis began in the early nineteenth century. The Russian physicist Alexander Reuss observed that when electricity was passed through a glass vessel containing a mixture of water and clay, the particles of clay migrated toward the positive pole. Reuss's discovery was developed with the work of Michael Faraday, in England, and E. DuBois-Reymond, in Germany. Independently, they demonstrated that, in an electric field, any negatively charged particles move toward the positive pole, and positively charged particles move toward the negative pole. Further, the rate of movement was proportional to the relative charge on the particles: The greater the charge, the faster the rate of migration. Electrophoresis could thus be applied as a means of separating macromolecules within a mixture, on the basis of their electrical charges.

This application of electrophoresis has been particularly important in the development of the field of biochemistry. Biological molecules such as proteins and nucleic acids are charged polymers, and migrate in an electric field. Protein biochemists have utilized electrophoretic procedures in determining size or charge differences between molecules at the level of a single residue. Complex mixtures of proteins can be separated into separate species, allowing for evaluation of individual components. The same principle, when applied to the resolution of DNA or RNA, has allowed separation of macromolecules that differ as little as 1 percent in their molecular weight.

Like any technique in science, electrophoresis has evolved as technology has developed. As the greater flexibility of gel separation became apparent, the use of paper as a medium of separation declined. In like manner, the use of starch gels has been superseded by materials such as agarose and polyacrylamide, with the greater adaptability for separation of molecules of different molecular weights. The inclusion of detergents such as sodium dodecyl sulfate within the gel solution has somewhat eliminated problems associated with differences in three-dimensional structure, since the action of molecular sieving depends in part on the shape of the molecule.

Much of future technology in the area will involve methods for greater resolution of molecules that exhibit minor differences in shape or charge. Thus, gradient gel electrophoresis, using a continuum of increasingly smaller pore sizes, will become more common. The application of immunological methods directed against specific structures, in combination with electrophoresis, will add a dimension of increasingly fine resolution at the molecular level.

Purification of macromolecules through electrophoretic techniques has been instrumental in the understanding of the molecular properties of biological polymers. The procedure can rightly be said to have revolutionized both protein and nucleic acid biochemistry.

Principal terms

AGAROSE: a polymer consisting of the sugar galactose and its derivative, 3,6-anhydro-1-galactose, commonly used as a gel

GEL: a cross-linked network of polymers that serves as a molecular sieve; gels generally consist of either agarose, dextran, starch, or acrylamide

ION: an electrically charged atom or group of atoms

MOLECULAR WEIGHT: the relative weight of a molecule, based on a scale in which the weight of an oxygen atom is expressed as 16

MOVING-BOUNDARY ELECTROPHORESIS: an analytical procedure in which the movement of the boundary between a mass of particles and the solvent in which they are immersed is measured as a function of time

PH: a term used to indicate acidity or alkalinity; pH values between 0 and 7 indicate acidity; pH values between 7 and 14 indicate alkalinity

POLYMER: a complex consisting of repetitive units of the same chemical elements

ZONE ELECTROPHORESIS: the migration of macromolecules through a solvent supported by a gel or paper strip; the sample is applied as a spot or thin layer on the gel or paper

Bibliography

Freifelder, David. PHYSICAL BIOCHEMISTRY. San Francisco: W. H. Freeman, 1982. An outstanding reference text in the area, which includes a comprehensive review on the biochemical use of electrophoresis. Examples and diagrams are abundant and provide a clear sense of the subject to anyone with a basic knowledge of science. A bibliography is also provided.

Gray, George. "Electrophoresis." SCIENTIFIC AMERICAN 183 (December, 1951): 45-53. Although this is an early article on the subject, it provides an excellent history on the development of electrophoretic principles. The description of procedures also provides an interesting perspective of laboratory science and its "hands-on" approach prior to the advent of advanced technology.

Hames, B. D., and D. Rickwood, eds. GEL ELECTROPHORESIS OF PROTEINS. Washington, D.C.: IRL Press, 1981. The editors have put together a comprehensive text on gel electrophoresis of proteins that provides an excellent review of the subject. Techniques are outlined, and many examples of applications are presented.

Lewis, Lena A., and Jan J. Oppit, eds. CRC HANDBOOK OF ELECTROPHORESIS. Boca Raton, Fla.: CRC Press, 1980. A good source book on the subject with excellent diagrams and bibliography. While basically a technical manual, the text does provide insight into procedures associated with electrophoresis.

Moody, G. J., and J. D. R. Thomas. PRACTICAL ELECTROPHORESIS. Watford, England: Merrow, 1975. Moody and Thomas present a good general description of the process of electrophoresis, emphasizing practical applications and minimizing theory. Examples are numerous, with discussion of both advantages and disadvantages of various techniques. The text also provides suggestions for handling problems that may arise.

Shaw, Duncan J. ELECTROPHORESIS. New York: Academic Press, 1969. Shaw presents an excellent introduction to the principles and applications of electrophoresis. While the book provides a basic account of these areas, a minimal knowledge of physics and chemistry would aid in the understanding of these phenomena. Formulations are numerous, and diagrams useful to the novice are minimal.

Smith, Ivor, ed. CHROMATOGRAPHIC AND ELECTROPHORETIC TECHNIQUES. New York: John Wiley & Sons, 1988. Smith provides an excellent discussion of the theory behind gel electrophoresis and outlines the basic protocols used for carrying out the process. Various methodologies used in separation techniques are described.

Solvation and Precipitation