Electrophoresis

Principle

Electrophoresis is the movement of an electrically charged substance under the influence of an electric field. This movement is due to the Lorentz force, which acts on the charge of the particle under study and which is dependent on the ambient electrical conditions. This force is given below by:

(1)

F (a vector, in the equation expressed by the dash above the F) is the Lorentz force, q is the charge (a scalar) of the particle, E is the electric field, a vector. The resulting electrophoretic migration is countered by forces of friction, such that the rate of migration is constant in a constant and homogeneous electric field:

F_f = vf , (2)

where v is the velocity and f is the frictional coefficient. Since in the stationary condition forces of friction and Lorentz force become the same, it holds that:

. (3)

The electrophoretic mobility μ is defined as:

μ= v/E = q/f . (4)

The expression (4) above applied only to charged molecules (ions) at a low concentration and in a non-conductive solvent. Poly-ionic molecules are surrounded by a cloud of counter-ions which alter the effective electric field applied on the molecule. This render the previous expression a poor approximation of what really happens in an electrophoretic apparatus.

Application

Electrophoresis is used as an preparative and analytical tool in molecular biology.

Gel electrophoresis is an application of electrophoresis in molecular biology, especially in DNA techniques. The gel-electrophoresis apparatus uses a positive and a negative charged pole. The (macro)molecule, e.g. DNA is loaded in on the negatively charged pole and pulled through the gel toward the positive pole. 

The charged of a DNA molecule is provided by negative phosphate groups. The content of the buffers (solutions) and gels used to enhance viscosity greatly affects the mobility of macromolecules. he gel used in the procedure is typically an agarose or a polyacrylamide gel, depending on the type of molecule studied. The thickness of the gel is typically ca. 8 mm.  Within the gel is a tightly woven matrix that the molecules must pass through as they are moving from one pole to the other. The smaller molecules can weave in and out of the matrix of the gel with more ease, compared with larger molecules. Wells, or rectangular openings, are formed along one edge of the gel. These wells mark the different lanes in which a sample may be loaded. Agarose is applied to separate large DNA fragments (100-50,000 base pairs) with modest resolution (some 50 base pairs). Polyacrylamide is useful for small fragments (10-1000 pairs) with single base-pair resolution.

Modifications are e.g. gradient (detergent) gel electrophoresis and (water cooled) rapid agarose gel electrophoresis.

More Info

The mobility depends on both the particle properties (e.g., surface charge density and size) and solution properties, e.g., ionic strength, electric permittivity, and pH. (permittivity describes how an electric field affects and is affected by a medium, e.g. air or water). For high ionic strengths, an approximate expression for the electrophoretic mobility μ_e is given by the equation,

μ_e = ε∙ε ₀∙ζ / η,

where ε is the dielectric constant (relative permittivity) of the liquid, ε₀ is the permittivity of vacuum, η is the viscosity of the liquid, and ζ is the zeta potential (i.e. surface potential) of the particle.

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