electrophoresis

1. ELECTROPHORESIS:
Definition: Electrophoresis is the migration of charged
particles or molecules in a medium under the influence of
an applied electric field.
‘Electrophoresis’ comes from Greek word meaning
transport by electricity’.
‘Electro’ refers to electron flow or current.
‘Phoresis’ refers to movement.
Thus Electrophoresis is movement under electric current

2. • Is a separation technique and is simple, rapid and highly sensitive
• used in clinical laboratories to separate charged molecules from each other
in presence of electric field
Proteins in body fluids: serum, urine
Proteins in erythrocytes: hemoglobin
Nucleic acids: DNA, RNA

4. Principle :
 the migration of charged particle of any size in
liquid medium under the influence of an electric
field.
Depending on kind of charge the molecule carry,
they move towards either to cathode or to Anode
An ampholyte become positively charged in acidic
condition and migrate to cathode, in alkaline
condition they become negatively charge and migrate
to anode

5. The rate of migration of an ion in electrical field depend
on factors,
1. Net charge of molecule
2. Size and shape of particle
3. Strength of electrical field
4. Properties of supporting medium
5. Temperature of operation
Size and shape of the particle decide the velocity with
which the particle will migrate under the given electrical
field and the medium.

7. Molecule in an electric field

8. The buffer in electrophoresis has twofold purpose:
Carry applied electrical current
They set the pH as which electrophoresis is carried out.
Thus they determine;
Type of charge on solute.
Extent of ionization of solute
Electrode towards which the solute will migrate.
The buffer ionic strength will determine the thickness of the ionic cloud.

9. Commonly used buffers;
Buffer pH value
Phosphate buffer around 7.0
Tris-Borate-EDTA buffer (TBE) around 8.0
Tris-Acetate EDTA buffer (TAE) above 8.0
Tris Glycine buffer (TG) more than 8.5
Tris -Citrate-EDTA buffer (TCE) 7.0
Tris -EDTA buffer (TE) around 8.0
Tris -Maleic acid -EDTA buffer (TME) around 7.5
Lithium Borate - buffer (LB) around 8.6

10. Supporting medium
• Supporting medium is a matrix in which separation
takes place, either on slab or capillary form.
Eg.: Starch gel , Cellulose acetate , Agarose ,
Polyacrylamide gel

11. What Factors are Affecting on Electrophoresis Methods
The rate of migration (Separation of particles) during electrophoresis will depend
on the following factors:
1. The Sample
2. The Electric Field
3. The Medium
4. The Buffer
1. THE SAMPLE:
a)Charge: The higher the charge, greater is the electrophoretic mobility.
The charge is dependent on pH of the medium.
b) Size: The bigger molecules have a small electrophoretic mobility compared to the
smaller particles.
c) Shape: The globular protein will migrate faster than the fibrous protein

12. 2. THE ELECTRIC FIELD:
The rate of migration under unit potential gradient is referred to as “Mobility of the
ion”. An increase in potential gradient increases the rate of migration.
3. THE MEDIUM:
The inert medium can exert adsorption & molecular sieving effects on the pa
rticle, influencing its rate of migration.
a)Adsorption: It means retention of a component on the surface of supporting
medium.
b) Molecular sieving:
The smaller molecules pass through the pores easily, but the larger molecules are
retarded.
4. THE BUFFER: The buffer can affect the electrophoretic mobility of the sample in
various ways.
- composition, PH, ionic strength

13. Classification of Electrophoresis
Electrophoresis is working on the basic principle of migration of charged
particles under the influence of electric field.
Electrophoresis
Free Electrophoresis
(or) Electrophoresis
without stabilizing
media
Zone
Electrophoresis (or)
Electrophoresis in
Stabilizing media

14. •FREE SOLUTION ELECTROPHORESIS:
•In this Electrophoresis, Stabilizing media (Agar, Starch, Polyacrylamide) is not using.
FREE SOLUTION ELECTROPHORESIS
MOVING
BOUNDARY
ELECTROPHORESIS
(M.B.E.):
FREE-FLOW
ELECTROPHORESIS:
MICRO
ELECTROPHORESIS:
CAPILLARY
ELECTROPHORESIS
(CE)
MICROCHIP
ELECTROPHORESIS
(MCE)

16. 1. MOVING BOUNDARY ELECTROPHORESIS (M.B.E.):
•Moving-boundary electrophoresis or free-boundary electrophoresis is
electrophoresis in a free solution.
•was developed by Arne Tiselius in 1937.
•The apparatus includes a U-shaped cell filled with buffer solution and
electrodes immersed at its ends.
•the motion of charged particles through a stationary liquid under the
influence of an electric field.
The sample applied could be any mixture of charged components like a
protein mixture.
•On applying voltage, the compounds will migrate to the anode or cathode
depending on their charges.

18. •At one end, the sample is injected at a defined spot, and at the other
end the fractions are collected through an array of tubings, which leads
to a 96-well microtiter plate.
•This is the only continuous electrophoretic separation method.
• this technique is also used for the identification, purification, and
isolation of cell organelles and membranes or whole cells such as
erythrocytes, leukocytes, tissue cells, the causal agent of malaria and other
parasites.

20. c MICRO ELECTROPHORESIS:
•It involves the observation of motion of small particles in an electric field
with a microscope.
•In modern days this technique is applied only for measuring the Zeta Potentials
of cell such as RBCs, Neutrophils and Bacteria etc.
if the zeta potential falls below a certain level, the colloid will aggregate due to the
attractive forces. Conversely, a high zeta potential maintains a stable system.
The apparatus includes a capillary cell, two chambers that include electrodes, and
a means of observing the motion of particles.
The apparatus is filled with very dilute suspension and the chambers are closed.
A direct-current voltage is applied between electrodes in the respective chambers.
One uses a microscope to determine the velocity of particles.
By making reasonable assumptions about the size of the observed particles and
the electrical conductivity it is possible to calculate the value of the zeta potential.
Zeta potential values near to zero indicate that the particles in the mixture are
likely to stick together when they collide, unless they also are stabilized by non-
electrical factors.

23. Capillary Electrophoresis (CE), also known as free solution capillary
electrophoresis.
•Capillary electrophoresis is a separation technique in which charged
species are separated, based on charge and size, by their different rates of
migration in an electric field.
•The capillary is made of negatively charged fused silica inner wall which
forms an electrical double layer with cations in the running buffer.
•These cations in the electrical double layer move towards the negative
cathode and hence the overall direction of flow also known as the
electroosmotic movement is towards the cathode.
•A mixture in a solution can be separated into its individual components
quickly and easily.
•The separation is based on the differences in electrophoretic mobility,
which is directed proportional to the charge on the molecule, and inversely
proportional to the viscosity of the solvent and radius of the atom.
The velocity at which the ion moves is directly proportional to the
electrophoretic mobility and the magnitude of the electric field.