Principle of Electrophoresis

General principle of Electrophoresis

Under the influence of the electric field, the migration of charged particles happens in the liquid medium. Depending on the charge carried by molecules, they move either towards anode or cathode. Ampholyte becomes negatively charged in basic conditions and migrates towards the anode. Also, the ampholyte gets a positive charge in acidic conditions and moves towards the cathode. For eg- Protein consists of ionizable carboxyl and amino groups. Ions moving towards cathode are cations and those moving towards anode are anions. Nucleic acid has a negative charge due to the phosphate backbone and so, it migrates towards the anode in DNA electrophoresis. But, protein will carry a negative or positive charge depending on solvent pH and isoelectric point.

An electrophoresis is an incomplete form of electrolysis because the electric field is removed before the molecules reach the electrode, yet the molecules have already been separated due to electrophoretic mobilities Then, the separated molecules are located with staining by a dye or autoradiography (for radioactive sample). Some electrophoresis processes are based on the charge of molecules, while some rely on the molecular size of molecules.

Factors Impacting Electrophoresis

There are numerous factors which impact the electrophoresis process. The rate of migration of biomolecules depend on the following factors:

• Molecule’s net charge
• Shape and size of particle
• Electric field’’s strength
• Supporting medium’s properties
• Operation temperature

• Electrophoretic velocity depends on both inherent factors and the external environment. Inherent factors are the magnitude of charge, charge density, molecular weight, and quaternary or tertiary structure (shape). The external environment consists of the pH of the solution, the electric field applied the viscosity of temperature and temperature. 
• Buffer serves two purposes - 
  1. Carrying of applied electric current
  2. Setting of pH at which process is carried out
• Thus buffers determine extent of solute ionization, charge on solute and direction of migration. They also determine the thickness of the cloud of ions.
• Supporting medium is a matrix (gel), in which separation of biomolecules takes place. It can be in slab or capillary form.
• The separation of molecules is dependent on the ratio of charge to mass of protein, pore size of medium and molecular size. Starch gel, cellulose acetate gel, polyacrylamide gel, and agarose gel are used as supporting media.

Charge of molecule

Depending on the net charge carried out by the molecule in the presence of an electric field, the molecule migrates towards anode or cathode.

Voltage

When a potential difference is applied across the electrodes, potential gradient is generated, which is the ratio of voltage and distance between the electrodes.

Voltage is the force, which drives the molecule towards the electrodes. The distance migrated is directly proportional to the time of passage of the electric field.

pH of Buffer

Based on the pH of the solvent, the particle carries charges since it is amphoteric. If there is a zwitterion, the overall charge is neutral, which will lead to zero mobility. Mobility is the ratio between the velocity of the ion and electric field strength. Mobility or movement or migration is directly proportional to the magnitude of charge possessed, which is dependent on the solvent’s pH. The pH of the solvent is maintained by the usage of buffers at different pH. The direction and speed of protein migration are influenced by buffer pH. 

As a function of the environment, protein’s net charge is determined as the sum of the electric charges present on the surface. If there are more positive charges, protein will migrate faster to the cathode. If there are excesses of negative charges, protein will move faster towards the anode. At the isoelectric point (pI) of a particular protein, the net charge in the protein is zero and it does not migrate in the electric field. The protein exhibits a net negative charge when the pH of the solvent is above pI and migrates towards the anode. When the pH of the solvent is below pH, the protein will show a positive charge and migrate towards the cathode.

Size and Shape of Particle

The shape and size of the particle decide the velocity, with which particles migrate towards the electrode. Shorter molecules move faster as they move out of the gel with ease, while longer molecules migrate slower to an electrode.

Charge and frictional coefficient determine the mobility of the particle. The higher the electric field, the migration of particles will be faster and migration to the target can be attained in shorter times. However, the frictional force due to viscosity and hydrodynamic size of the molecule retards the movement.

Ionic Strength of Buffer

The charge carried by the protein is influenced by the ionic strength of the buffer. At the lower ionic strength of the buffer, proteins carry more charge, and hence, faster migration takes place. When the ionic strength of the buffer is higher, the proteins carry a lesser charge and the migration becomes slower. Also, lower ionic strength leads to higher resistance with the lower current generation, leading to lesser heat generation. Hence, the lower ionic strength of the buffer is preferable. But, there is a chance of poorer separation of molecules.

Heat generation is one of the problems in the electrophoretic process. Power generated in the process is often dissipated as heat. Thermally labile samples are sensitive to heat. Also, an increase in the diffusion of samples leads to sample broadening. A stabilized power supply is the need of the hour to eliminate heating fluctuations and provide constant power.

Voltage Gradient

If there is more voltage gradient present in the electric field, protein will migrate towards the electrode at a faster rate. 

Electro-osmosis

A liquid’s relative motion in a solid medium in an electric field is electro-osmosis. Electro-osmosis is involved in distorting the sample stream, limiting the separation of molecules. There is poor resolution in paper electrophoresis as the paper has a negative charge and the buffer has a positive charge. This distorts the flow and the sample’s migration will change.

Conclusion

.The principle of electrophoresis is mainly based on the electrokinetic mechanism. Many factors influence the efficiency of the process. Some parameters will be suitable for some samples and some types of electrophoresis. Some variables are compatible with some type of gels. Compromise without affecting the quality of the process by a thorough understanding of the principle is the need of the hour.