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2what electrophoresis is
2The components of the system.
2Types of electrophoresis.
2What capillary electrophoresis is.
2Separation principle of CE.
2What CE instrumentation is comprised of.
2What CZE and MECC are and what they are used
for.
 

 
3

Electro=charge , phoresis=carry
It¶s a separation of charged molecules by difference in their rate of migration in an
electric field.

Electrophoresis is a technique wherein differential movement of charged species (ions)

occurs under the influence of an electric field.
The technique was introduced be Tiselius in 1973 when protein mixtures were placed
between buffer solutions in a tube and an electric field was applied. The result of
this experiment was that sample components migrated in a direction and at a rate
determined by their charge and mobility. (this work was awarded a noble prize).
Separation efficiency in the early electrophoresis experiments were limited by thermal
convection and diffusion so experiments were conducted instead in anti-convective
media such as polyacramide or agarose gels in either slab or tube formats.
  


 


2 Molecules to be separated. (protein-nucleic acids)

2 Support medium.
2 Buffer system ,electrolyte, have ionic molecules to be able to
have conductivity.
2 Power source.
 


 

2 Paper electrophoresis, somehow similar to TLC

technique.
2 Gel electrophoresis.
2 Capillary electrophoresis, the latest technique.
 D

 
Gel electrophoresis has long been a technique used for separating biological
macromolecules such as nucleic acids and proteins, however the technique does
suffer with long analysis time, low efficiencies and limitation with respect to
detection and automation.
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The latest and the most important technique.

Capillary electrophoresis (CE) employs narrow bore capillaries
(typically 20-200 µm i.d) filled with an electrolyte solution
across which a voltage is applied in order to perform high
efficiency seperations of both large and small analyte ions.
Separations are facilitated by the use of high voltages which may
generate electro-osmotic and electrophoretic flow of both the
electrolyte solution and analyte ions respectively.
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In a typical electrophoretic separation, the capillary first filled with electrolyte, next,
sample introduced to capillary end which is away from the detector (usually Anode
end of the capillary).
The ends of the capillary are then dipped in reservoirs containing electrodes and
electrolyte.
One electrode is connected to cable leading to a high voltage output, and the other (at
the detector end) connected to earthing cable.
Subsequent apply to the voltage (typically 5-30 kV) across the capillary causes
migration of the electrolyte and analyte toward the detector.
Plot of the detector response against time will generate and known as
electropherogram.
Migration rate, or mobility, of the solute under the applied field, largely governed by
their size and charge (size to charge ratio).
The small positively charge analyte migrate ahead of larger analyte of the same
charge,and vice versa for negatively charged solutes moving toward the anode.
Ionic mobility (µe) is related to charge/size ratio as shown:
 


 


‰

where:
‰ ionic mobility or electrophoretic.
charge.
 viscosity of solution.
radius of ion
‰ unit is (m²s
 
 


  

The actual electrophoretic velocity of the ions is related to their mobilities and
the magnitude of the electric field.

å
‰

 
å ionic velocity
‰ ionic mobility

electric field applied
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 .
2 MW molecular size
2 Molecular shape
2 Molecular charge.
    

 
2 Electric field strength (V/cm)
2 Porosity of the support medium (â 
2 


   
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2 Mobility is proportional to charge/MW

-charge is effected by buffer pH.
2 Mobility is proportional to field strength.
2 Mobility is inversely proportional to buffer conductivity.