Electrophoresis is one of the important tool in Diagnostic labs, Pharma labs, Forensic labs and many more labs. First of all we should know What is Electrophoresis? Electrophoresis is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.
This electro-kinetic phenomenon was observed for the first time in 1807 by Ferdinand Frederic Reuss (Moscow State University), who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. This method was developed by “Arne.W.K.Tiselius” in 1937
It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid.
Factors affecting on Electrophoresis:
The rate of migration (Separation of particles) during electrophoresis will depend on the following factors:
1. The Sample
2. The Electric Field
3. The Medium
4. The Buffer
1. The Sample:
Charge/mass ratio of the sample dictates its electrophoretic mobility. The mass consists of not
only the size (molecular weight) but also the shape of the molecule.
a) Charge: The higher the charge, greater is the electrophoretic mobility. The charge is dependent on pH of the medium.
b) Size: The bigger molecules have a small electrophoretic mobility compared to the smaller particles.
c) Shape: The globular protein will migrate faster than the fibrous protein
2. The Electric Field:
The rate of migration under unit potential gradient is referred to as “Mobility of the ion”. An increase in potential gradient increases the rate of migration.
3. The Medium:
The inert medium can exert adsorption & molecular sieving effects on the particle, influencing its rate of migration.
a) Adsorption: It means retention of a component on the surface of supporting medium. The rate and resolution of the electrophoretic separation can be efficiently reduced by adsorption.
b) Molecular sieving: Media such as “Polyacrylamide”, “Agar”, “Starch” & “Sephadex” have cross-linked structures giving rise to pores within the gel beads.
- Sephadex, molecules larger than the pores are excluded from entering the gel beads & these molecules migrate faster.
- Polyacrylamide, Starch & Agarose the larger molecules also are made to squeeze through the pores. The smaller molecules pass through the pores easily, but the larger molecules are retarded.
4. The Buffer: The buffer can affect the electrophoretic mobility of the sample in various ways.
A) Composition: Commonly used buffers are “Formate”, “Acetate”, “Citrate”, “ Phospahate”, “EDTA”
“Acetate” “Pyridine”, “Tris” (2-amino, 2-hydroxymethyl, 1,3-diol pentane) and “Barbitone” etc.
The choice of buffer depends upon the type of sample being electrophoresed.
b) Ionic Strength: “Ionic Strength (I) is a measure of the electrical environment of ions in a sol”. When increase ionic strength of the buffer means a larger share of the current being carried by the buffer ions & meager (small quantity) proportion carried by the sample ions.
When decrease ionic strength, a larger share of the current being carried by the sample ions leading to a faster separation.
Note: The ionic strength used is usually between 0.05 to 0.1M.
The pH determines the degree of ionization of organic compounds; it can also affect the rate of migration of these compounds. When increase pH, increases ionization of organic acids. Decrease in pH, increases ionization of organic bases. E.g.: an Ampholyte (Amino acid) - The amino acid has both acidic & basic properties
Read More: Chromatography, types, Principle and Applications.