Electrophoresis is a physical method used to separate charged particles like proteins and amino acids using an electric field. It works by applying a voltage across a medium like paper or gel containing the sample, causing charged molecules to migrate at different rates depending on factors like their size, shape, and charge. The history of electrophoresis began in 1931 with Arne Tiselius' work separating proteins. It continues to be developed today and has applications in biochemistry like separating amino acids, proteins, enzymes, and antibiotics.

1. Electrophoresis
Dr.S.H. H. Burungale

2. Electrophoresis
The history of electrophoresis for molecular
separation and chemical analysis began with the
work of Arne Tiselius in 1931, while new separation
processes and chemical analysis techniques based on
electrophoresis continue to be developed in the 21st
century.

3. CONTENT
Introduction
Principle
Factors affecting
Conventional electrophoresis
General operation
Technical and practical
Consideration
Types of electrophoresis

4. THEORY OF ELECTROPHORESIS
INTRODUCTION: Electrophoresis is a physical
method of analysis which involves separation of the
compounds that are capable of acquiring electric charge
in conducting electrodes.
DEFINITION: • Electrophoresis may be defined as the
migration of the charged particle through a solution
under the influence of an external electrical field.
• Ions that are suspended between two electrodes tends
to travel 3 towards the electrodes that bears opposite
charges

5. Principle :
Comprehensive term that refers to 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 toAnode
An ampholyte become positively charged in acidic condition and migrate to cathode,
in alkaline condition they become negatively charge and migrate to anode.

6. E.g.. as protein contain the ionizable amino and carboxyl group.
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
1. Mobility Under the electrical field, the mobility of the particle is determined by two
factors: Its charge Frictional coefficient Size and shape of the particle decide the
velocity with which the particle will migrate under the given electrical field and the medium.

7. 2. Strength of electrical field It determined by the force exerted on the particle, and the charge
the particle carrying. F=QV when force is exerted on the particle it start moving, however the
moment is restricted by the experience of the frictional force because of the viscosity.
3. Effect of pH on Mobility
As the molecule exist as amphoteric , they will carry the charges based on the solvent pH.
Their overall net charge is NEUTRAL when it is at zwitter ion state.And hence the mobility is
retarded to zero.
•Mobility is directly proportional to the magnitude of the charge, which is functional of the pH
of solvent.
•The pH is maintained by the use of Buffers of different pH.

8. TYPES OF ELECTROPHORESIS
1) Zone Electrophoresis
a) Paper Electrophoresis
b) Gel Electrophoresis
c) Thin Layer Electrophoresis
d) Cellulose acetate Electrophoresis
2) Moving Boundary Electrophoresis
a) Capillary Electrophoresis
b) Isotachophoresis
c) Isoelectric Focussing

10. ZONE ELECTROPHORESIS
It involves the migration of the charged particle on the supporting media.
Paper, Cellulose acetate membrane, Starch Gel, Poly acrylamide.
Components separated are distributed into discrete zone on the support media.
Supporting media is saturated with buffer solution, small volume of the sample is applied as
narrow band.
On application of PD at the ends of a strip components migrates at a rate determined by its
electrophoretic mobility.
ADVANTAGES: Useful in biochemical investigations. Small quanity of sample can be
analysed. Cost is low and easy maintenance.
DISADVANTAGES: Unsuitable for accurate mobility and isoelectric point determination. 5
Due to the presence of supporting medium, technical complications such as capillary flow, electro
osmosis, adsorption and molecular sieving are introduced.

11. GENERAL METHOD OF OPERATION
Saturation of the medium with the buffer.
Sample application. Electrophoretic separation.
Removal of the supporting media.
INSTRUMENTATION
Electrophoretic chamber.
Electrodes.
Diffusion barriers.
Supporting/ Stabilizing media. (inert to sample and to any developing reagents).

13. SAMPLE
Charge : Rate of migration increases with increase in net charge.
It depends on pH.
Size : Rate of migration decreases for larger molecules. It is due to
increase frictional and electrostatics forces.
Shape : Molecular have similar charge but differ in shape exhibit
different migration rate.
ELECTRIC FIELD According to ohms law I=V/R Current =
Voltage / Resistance
Voltage : Increase in voltage leads to increase in rate of migration
Current : Increase in current leads to Increase in voltage, so the
migration also Increases
Resistance: If resistance increase migration decreases.

15. IONIC STRENGTH As ionic strength of buffer increases
Proportion of current carried by buffer increases
Proportion of current carried by the sample decreases and hence showing decrease in
sample rate of migration.
High ionic strength
• Also increases overall current and hence heat is produced
As ionic strength of buffer decreases
Proportion of current carried by buffer decreases
Proportion of current carried by the sample increases and hence showing increase in
sample rate of migration.

16. pH
pH determines the ionization, if ionization of organic acid
increases as pH increases, ionization of organic acid
decreases as pH decrease.
Therefore the degree of ionization is pH dependent.
SUPPORTING MEDIUM Adsorption
Adsorption is the retentio n of sample molecule by
supporting medium.
Adsorption causes tailing of sample so that it moves in the
shape of a ‘comet’ rather than a distinct compact band
Adsorption reduces both the rate of migration and
resolution of separation of molecule.

22. Applications- Separation of amino acids into acidic or basic type Separation
of proteins in serum (into albumin alpha1, alpha2, beta and gamma
globulins). Study of Sickle Cell Anaemia in Hemoglobin Separation of
enzymes in blood. Separation of antibiotics in different samples can be
carried out
SEPARATION OF AMINO ACIDS AT NEUTRAL SOLUTION: The acidic amino
acids will have a net positive charge and will migrate toward the cathode(-
ve) The basic amino acids will have net –ve charge and migrate toward the
anode(+ pole). Electrically neutral amino acids or the zwitter ion will not
migrate and they will stay at the origin. Ninhydrin is added on the spot of
amino acids.

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