Capillary Electrophoresis
In capillary electrophoresis process, a capillary
tube is used. It separate mixture components on the basis of
molecular weight and charge.
Fused Silica Capillary tube:
It is made up
of fused silica. The external diameter is 75-100µm and internal diameter is
50-75µm. The length of capillary tube is 25-100cm.
A jacket of
insulator is present outside the capillary tube. The gel is filled in the
capillary tube which is highly purified. Gel may be made of polyacrylamide,
starch or cellulose acetate.
It is more efficient then gel electrophoresis.
Bare
silica electrophoresis:
It is known as uncoated capillaries. It is made up
of highly purified fused silica.
This contain silanol group Si-OH which shows
different behavior with the change of pH.
Types of Capillary Electrophoresis:
The wall of Capillary electrophoresis is negatively
charged. Two mechanisms occur in the capillary tube:
Electro-osmotic flow
Electrophoretic mobility
Electro-osmotic flow
It is the movement of separation of mixture
components through the silica capillary as the result of existence of zeta
potential at the solvent silica interference. It explains the flow of buffer
solution.
The potential difference exists between surfaces
of solid particles immersed in conducting liquid and bulk of liquid.
Capillary wall contain negative charge due to basic
pH. Some of the negative charge is neutralize by the attraction of cation and
form fixed layer.
Now fix layer having negative charge and
concentration of cation is high as compare to concentration of anion make a
layer known as diffused layer.
The flow of diffused layer and bulk solution is
always from anode to cathode because the concentration of cation is higher
than the anion .
Diffused layer and fixed layer is collectively
called electrically double layer.
Electrophoretic Mobility
It is represented by µeq. It is
responsible for the separation of mixture components on the basis of charges. The
velocity by which solute moves under the influence of electric field, this
velocity is called electrophoretic velocity veq.
vep = µeq E
Where µeq is the electrophoretic mobility
of solute and E is the magnitude of electric field. The electrophoretic
mobility of solute is defined as:
µeq =
q / 6
where q is the charge on solute, Æž is the viscosity of buffer
solution and r is the stokes radius of solute. Following
charges are present in the bulk solution:
Negative charges moves towards anode.
Positive charges move towards cathode.
Neutral particles cannot move.
Dependence of electrophoretic mobility:
· Ionic charge on analyte
· Frictional retardation force on analyte
Suppose we have two molecules having same size, the
molecules which have greater charge will move faster as compared to the other
molecule. So that electrophoretic
mobility is directly proportional to charge concentration of the molecule.
Electrophoretic
mobility µep is inversely proportional to the frictional
retardation. If the friction is high, the sample
molecules show slow movement.
Frictional
retardation force on analyte depends upon following factors:
Size of molecule
If the size of molecule
is small, it shows quick movements towards their respective electrodes so that
their electrophoretic mobility is high.
If the size of molecule
is large, their velocity is less as compared to small size molecules so that
electrophoretic mobility is less.
Shape
of molecules
Regular shape molecule bears less friction so that
electrophoretic mobility is high.
Irregular shape molecules bear more friction so that
their electrophoretic mobility is less.
Stokes Radius
The size of ion in electrophoresis is its stokes
radius.
0 Comments