ELECTROPHORESIS

Seat no- 1917268

HISTORY OF ELECTROPHORESIS
:
• In 1930,Arne Tiselius developed Moving
boundary electrophoresis.

• Tiselius was awarded the 1948 Nobel Prize in

chemistry for his work on the separation of
colloids through electrophoresis, the motion of
charged particles through a stationary liquid
under the influence of an electric field.

• Moving-boundary electrophoresis (MBE also free-

boundary electrophoresis) is a technique for
separation of chemical compounds by
electrophoresis in a free solution
 •The method spread slowly until the advent of effective zone of
electrophoresis methods in the 1940s and 1950s, which used
filter paper or gels as supporting media.

•By the 1960s, increasingly sophisticated gel electrophoresis

methods made it possible to separate biological molecules
based on minute physical and chemical differences, helping to
drive the rise of molecular biology.
 DEFINITION
:
 Electrophoresis may be defined as the migration of
the charged particles through a solution under the
influence of an external electric field
 Ions that are suspended between two electrodes
tends to travel towards the electrodes that bears
opposite charges.
 PRINCIPLE
• When charged molecules are placed in an electric field, they
:
migrate towards either the positive or negative pole according
to their charge. In contrast to proteins, which can have either a
net positive or net negative charge, nucleic acids have a
consistent negative charge imparted by their phosphate
backbone, and migrate toward the anode.
•The separation effect on the ionic particles results from differences
in their velocity (v), which is the product of the particle's mobility
(m) and the field strength (E):
v=mxE

•The mobility (m) of an ionic particle is determined by particle size,

shape, and charge, and the temperature during the separation, and
is constant under defined electrophoretic conditions.
FACTORS AFFECTING
ELECTROPHORESIS
A] Sample-
1) Charge : Rate of migration increases with increase in net
charge.It depends on pH.

2) Size : Rate of migration decreases for larger molecules.It is

due to increase frictional and electrostatic forces.

3)Shape : Molecules have similar charge but differ in shape

exihibit different migration rate.
B]Buffer-
1)Composition
2)pH
3)Concentration
4)Heat generation in electric field
C]Electric field-
According to ohms law, I=V/R

1)Voltage :Increase in voltage leads to increase in rate of

migration.

2)Current :Increase in current leads to increase in

voltage,so the migration also increases.

3)Resistance:If resistance increases migration decreases.

D]Supporting Medium-
1)Adsorption
2)Electroendo-osmosis
3)Molecular Sieving
TYPES OF ELECTROPHORESIS :
ZONE
ELECTROPHORESIS
PRINCIPLE Any electrophoretic technique
in which components are
separated into zones or bands
in a buffer, and stabilized in
solid, porous, or any other
support medium–eg, filter
paper, agar gel, or
polyacrylamide gel
COST Low and easy maintenance
ADVANTAGES 1)Useful in biochemical
investigations
2)Small quantity of sample
can be analysed
MOVING BOUNDARY
ELECTROPHORESIS
The moving boundary methos
allows the charged species to
migrate in a free moving
solution without the supporting
medium.
Expensive
1)Biologically active fractions
can be recovered without the
use of denaturing agents.
2)A reference method for
measuring electrophoretic
mobilities.
3)Minute concentrations of
DISADVANTAGES 1)Unsuitable for accurate 1)Elaborate
mobility and isoelectric optical
point determination. system are required.

2)Due to the presence of

supporting
medium,technical
complications such as
capillary flow,electro-
osmosis,adsorption and
molecular sieving are
introduced.
APPLICATIONS 1)Analysis of complex 1)Used to study the
biological mixtures behavior of a molecule in
2)To study homogenecity of an electric field.
a macromolecular system.

TYPES 1)Paper 1)Capillary

2)Gel 2)Isoelectric focusing
3)Thin Layer 3)Immuno electrophoresis
Electrophoresis 4)Isotachophoresis
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.
Gel electrophoresis - is a widely used type of electrophoresis in
which molecules are separated by movement through a porous
gel under the influence of an electrical field. The two main gel
materials are agarose and polyacrylamide. Gel electrophoresis is
used to separate nucleic acids (DNA and RNA), nucleic acid
fragments, and proteins.
Immunoelectrophoresis - Immunoelectrophoresis is the
general name given to a variety of electrophoretic techniques
used to characterize and separate proteins based on their
reaction to antibodies.
Capillary electrophoresis - Capillary electrophoresis is a type
of electrophoresis used to separate ions depending mainly on
the atomic radius, charge, and viscosity. As the name
suggests, this technique is commonly performed in a glass
tube. It yields quick results and a high resolution separation.

Isoelectric focusing - Isoelectric focusing (IEF or

electrofocusing) is a form of electrophoresis that separates
molecules based on different isoelectric points. IEF is most
often performed on proteins because their electrical charge
depends on pH.
REFERENCES :

1)https://www.slideshare.net/mobile/SayantiSau/techniques-of-
electrophoresis
2)https://www.slideshare.net/mobile/Pharmacistprabin/electrophore
sis-45407760
3)https://www.pharmatutor.org/pharma-analysis/explain-
electrophoresis-its-principle-and-factors-governing-it
4)https://en.m.wikipedia.org/wiki/History_of_electrophoresis
5)https://www.pharmatutor.org/pharma-analysis/explain-
electrophoresis-its-principle-and-factors-governing-it

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