Electrophoresis 



•   The migration of charged particles or molecules in a medium under the influence of an applied electric field.

•    The rate of migration of charged molecule depends on

•       The strength of electric field, size and shape.

•       Ionic strength and temperature of the buffer.

•       Molecular size of the taken molecule.

•       Net charge density of the taken bio molecule.

•       Shape of the taken bio molecule.

  • In the process of electrophoresis large molecules have more difficulty in moving through the supporting medium (i.e., gel)
  • Whereas the smaller medium has more mobility through it.


History

•       This technique was unknown until 1930.

•       The technique was invented by A-Tiselius

•       And he won Nobel prize in 1948.





Electrophoretic Mobility    

•   Electrophoretic mobility is defined as the rate of migration (cm/sec) per unit field strength (Volts/cm)

                              µ₌Q/6πɼȠ

•   Where            µ__ Electrophoretic mobility.

                           Q__ Net charge on the ion.

                            ɼ__ Ionic radius of the solute.

                            Ƞ__ Viscosity of the medium.

         For DNA: minimum 40 mins,    100-120 volts

         FOR PROTEIN: minimum 20 volts___ overnight.

•   The different components in a mixture will have different electrophoretic mobilities and hence they can be separated.

•   Mixtures of amino acids , proteins and nucleotides can be separated by their migration in an electric field .


                                                      


Electrophoresis Apparatus

Electrophoresis apparatus consists of –

  •    Buffer tank – to hold the buffer
  •    Buffer  
  •    Comb (SDS Page)
  •    Electrodes – made of platinum or carbon
  •    Power supply
  •    Support media



Types of electrophoresis







1)Paper Electrophoresis:

• Paper Electrophoresis is one of the zone electrophoresis.

• Paper of good quality should contain at least 95% α- cellulose and should have only a very slight adsorption capacity.

• 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 strips are immersed into buffer reservoirs containing the electrodes.






 


• The two tank will have same buffer

• Sample will be pour at specific position usually at the center of paper,( high voltage is applied and the spots migrate according to their charges).

• Otherwise pouring the sample on other place other than center will not be separated.

• After electrophoresis the separated components can be detected by a variety of staining technique.

• Time : 16-18 hours

• Now a days Ethidium bromide is used.




Applications

• Serum analysis for diagnostic purpose is routinely carried about by paper electrophoresis.

• Muscle proteins, egg white proteins, milk proteins and snake, insect venom analysis done by this technique.

• The chance for the wastage of sample is decrease by acetone, ethanol they are fixed up to solid support it enhance attachment.


ADVANTAGES

•       It is used for separation purpose

•       Diagnostic purpose

•       Various proteins analyzation

•       Easy to use and of low cost

DISADVANTAGES

•       Time consuming

  • Greater chances of impurity



2)Capillary electrophoresis:

•  The technique which use to separate ion base on their electrophoretic mobility

                 OR

• The technique in which we separate chemicals based on their charge to mass ratio

• Two terms are very important

             Electrophoretic mobility

             Electric osmotic force (EOF).


Electrophoretic mobility

  • Electrophoretic mobility: rate of movement per unit field strength

                                                          µ₌Q/6πɼȠ

•       Where     µ__ Electrophoretic mobility.

                           Q__ Net charge on the ion.

                            ɼ__ Ionic radius of the solute.

                            Ƞ__ Viscosity of the medium.

•       Movement is directly related with field strength

•       In electrophoresis a thin filament of saluryte fix with silica is used.


Electro osmotic force

•   The saluryte capillary tube have a negative charge inside.

•   When an analyte is put there is attraction between the walls as well as negative electrode

•    So a diffusion surface is produce refer as EOF

•    When a charge is positive it will move faster than EOF.

•    When a charge is negative it will move slower.



 

The instrumentation

•       Voltage power supply

•       Capillary tube __ saluryte

•       Sample__ analyte

•       Electrodes__ (anode +ive) ( cathode –ive)

•       Detector

•       U-V source

•       Output device.



Diagrammatically representation:

 

Application

  •   Used to separate
  •  Amino acids
  •  peptides
  •  proteins
  •  DNA fragments
  •  Nucleic acid
  •  Drugs/ even metals
  • Haemoglobinopathy screening
  • Multiple myeloma testing
  • Monitoring chronic alcoholism.

  


Advantages

•       No mass transfer between stationary or mobile phase.

•       Less labouries

•       Less time consuming

•       Only small amount of sample is required

•       High separation efficiency





DISADVANTAGES

  • Denser molecule hard to separate .

                                                                



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