Electrophoresis is one of the main techniques used for molecular separation for analysis of biological or other polymeric samples.

It is easy to perform and cost-effective and yet provides a high separation efficiency. It is mainly applied for analytical purposes.

Electrophoresis is widely used in biological and biochemical research, forensic medicine, veterinary science, clinical investigations, protein chemistry, food control, and molecular biology.

General Principle

Electrophoresis was originally used to analyze and separate proteins but is now applied to various chemical mixtures.

The fundamental principle behind the process of electrophoresis is the existence of charge separation between any surface and fluid in contact with it.

Surface carries an immobilized charge and the electrolyte in contact with the charged surface balances electric charge with an increased density of ions of the opposite charge.

Factors Affecting the Movement of the Particles

The process involves movement of electrically charged particles in a fluid under the influence of an electric field where the charged particles migrate in the direction of electrode bearing opposite charge.

Different molecules have different rate of migration because of difference in their charges and masses. The electrophoretic mobility of a particular molecule is its characteristic parameter.

It is dependent upon

  • pKa values of the charged groups
  • temperature
  • field strength
  • nature of the support material
  • type, concentration and pH of buffer used

Electrophoresis can be carried out in one-dimension or two-dimensions.

One-dimensional electrophoresis is routinely used to carry out separation of proteins and nucleic acids, where as two-dimensional separation of proteins is used for DNA fingerprinting.

Method Involved

Electrostatic columbic force exerts external electric field on the dispersed particles having an electric surface charge developing electric potential at an interface between two immiscible phases.

These two electric layers of charge are called electric double layer.

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 (Figure 1).

Figure 3. Diagrammatic representation of  process of electrophoresis
Figure 3. Diagrammatic representation of  process of electrophoresis

Kenneth A. Ferguson published Ferguson plot  for the analysis of hormones of the pituitary gland in starch gels.

The Ferguson plot is the semi-logarithmic plot of electrophoretic mobility versus gel concentration. According to the plot,

Mobility = migration velocity (cm/s) / field strength (V/cm)

There are three main types of electrophoretic separation methods, namely Zone electrophoresis (ZE), Isotachophoresis (ITP), and Isoelectric focusing (IEF). These methods may be used alone or in combination to separate molecules on both an analytical as well as preparative scale.

Separations in these modes are based on the difference in physical properties of the molecules in a mixture.

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