REVIEW ON ELECTROPHORESIS TECHNIQUES
Final Year Graduate student
Sri Lakshmi Narasimha College of Pharmacy,
Palluru, Chittoor-517132, Andhra Pradesh, India
Electrophoresis is also called as cataphoresis. It is the motion of dispersed particles relative to a fluid under the influence of spatially uniform electric field. This electro kinetic phenomenon was observed for the first time in 1807 by Reuss (Moscow State University), who noticed that the application of a constant electric field caused clay particles dispersed in water to migrate. It is ultimately caused by the presence of a charged interface between the particle surface and the surrounding fluid. Electrophoresis is a rapid separation technique.Factors governing the migration of ions are Charge of ions, Size of the ions, Viscosity of the medium, Voltage applied, pH of buffer and ionic strength. Factors affecting electrophoresis are Molecular characteristics (size, shape), Buffer Properties, Electric Field Characteristics, Temperature.
Reference Id: PHARMATUTOR-ART-1617
FACTORS GOVERNING THE MIGRATION OF IONS:
1. Charge of ions
2. Size of the ions
3. Viscosity of the medium
4. Voltage applied
5. pH of buffer and ionic strength
1. Charge of ions:
The electrophoretic mobility is directly proportional to the charge of the molecule, which means that mobility of a molecule is higher when its charge is higher.
2 Size of the ions:
The electrophoretic mobility is inversely proportional to its size, i.e.the mobility is more, when the size is less and vice versa. The mobility is also depends on the shape of the molecule.
3. Viscosity of the medium:
The electrophoretic mobility is directly proportional to the viscosity of the medium.
4. Voltage applied:
Higher the voltage applied faster the separation and sharp bands are obtained. However steps have to be taken to prevent evaporation of the solvent / buffer due to the heat generated by high voltage.
5. pH of Buffer and ionic Strength:
The pH of the buffer solution is to be used is depends on the nature of the components to be separated, in which they can dissociate into ions. Migration of compounds is inversely proportional to the ionic strength. At low ionic strength, migration is faster, but the separated bands appear diffuse. Usually ionic strengths (IS) of 0.05-0.5 is used in more separation.
FACTORS AFFECTING ELECTROPHORESIS:
Electrophoresis is a phenomenon, where charged particles migrate in the presence of an electric field. Biologists use electrophoresis to separate and identify biological molecules such as proteins, nucleic acids and amino acids that exist as either anions or cations. Several factors which affect the electrophoresis are
A) Molecular characteristics (size, shape)
B) Buffer Properties
C) Electric Field Characteristics
II. TYPES OF ELECTROPHORESIS:
1. Paper Electrophoresis
2. Affinity Electrophoresis
3. Capillary Electrophoresis
5. DNA Electrophoresis
6. Gel Electrophoresis
9. Immuno electrophoresis
11. Protein Electrophoresis
12. Pulsed Field Gel Electrophoresis
1. Paper Electrophoresis:
This technique is useful for the separation of small charged molecules such as amino acids and small proteins. A strip of filter paper is moistened with buffer and the ends of the strip are immersed into buffer reservoirs containing the electrodes. The samples are spotted in the centre of the paper, high voltage is applied and the spots migrate according to their charges. After electrophoresis, the separated components can be detected by a variety of staining techniques, depending upon their chemical identity.
This method also useful for determination of protein isoelectric point.It has very useful application in blood cell separation.
2. Affinity Electrophoresis:
This method include
A) Mobility Shift Electrophoresis
B) Charge Shift Electrophoresis
C) Affinity Capillary Electrophoresis
The methods are based on changes in the ectrophoretic pattern of molecules through bio specific interaction or complex formation. This method has been used for determination of a constants. Affinity electrophoresis may be used as an alternative quantification of the proteins.
3. Capillary Electrophoresis:
It is also known as Capillary Zone Electrophorsis (CZE). This can be use to separate ionic species by their charge and frictional forces and hydrodynamic radius. It can be performed in a capillary format. A typical system consists of two reservoirs and a capillary filled with a buffer solution. A high voltage is applied across the capillary by using a high-voltage power supply. The very small diameter capillaries (typically 5-100 µm) employed in this technique allow for efficient heat dissipation.
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