ELECTROPHORESIS.

Presented by:- Puneet Nirmal, Geeta & Haamid.

M.Pharm (Q.A.)
ISF College Of Pharmacy, Moga.

Submitted to:- Dr. Pooja Chawla.

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
towards the electrodes that bears opposite charges.
 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.
 It is a type of protein separation method which relies on
protein sizes to segregate the mixture.
 It is one of the highly efficient techniques of analysis and
sole method for separation of proteins for western blot,
RNA studies etc.
 But, on negative side it also time-consuming, expensive
and technical skilled procedure due to which is less
preferred in health care
 Electrophoresis is similar to other separation techniques
like chromatography but it differs in-terms of the types of
samples analyzed, the method used for separation,
principle used etc.
ELECTROPHORESIS & ITS PRINCIPLE
 Electrophoresis is a method of separation where in charged
molecules migrate in differential speeds in an applied electric
field.
 when electricity is applied to the medium containing biological
molecules, depending on their net charge and molecular size,
they migrate differentially, thus different proteins/DNA can be
separated.
 Depending on the kind of charge the molecule carry, they move
towards either to
*To cathode
* or anode
 FREE ELECTROPHORESIS

 In this type of electrophoresis a free electrolyte is taken in place

of supporting media.

 It is mostly of two types-

i. Micro Electrophoresis : It is mostly used in calculating Zeta
potentials(a colloidal property of cells in a liquid medium)of
the cells.
ii. Moving boundary Electrophoresis which for many years had
been used for quantitative analysis of complex mixtures of
macromolecules , esp. Proteins.
 ZONE ELECTROPHOROSIS
 It involves the migration of the charged particle on the supporting media caan be
Paper, Cellulose acetate membrane, Starch Gel, Polyacrylamide.
 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.

ADVANTAGES:
 Useful in biochemical investigations.

 Small quantity of sample can be analysed.

 Cost is low and easy maintenance.

DISADVANTAGES:
 Unsuitable for accurate mobility and isoelectric point determination. Due to the
presence of supporting medium, technical complications.
PAPER ELECTROPHOROSIS
 Paper Electrophoresis is one of the type of zone
electrophoresis.

Principle:
When charged molecules are placed in an
electric field, they migrate toward 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 towards the anode.
 EQUIPMENTS:
The equipment required for electrophoresis consist a basically
of two items, a POWER PACK and ELECTROPHORETIC
CELL.

1. Power pack: Power pack provides a stabilized direct

current & has controls for both voltage & current out put,
which have an out put of 0 to 500V and 0 to 150mA are
available.
2. The Electrophoretic cell: It contains: the electrodes,
buffer reservoirs, a support for paper and a transparent
insulating cover. The electrodes are usually made of
platinum.
WORKING.

1) A long strip of filter paper is moistened with a suitable

buffer solution of the desired p H and the sample is applied
transversely across the central part of the strip.
2) Ends are fixed to dip in buffer solutions in two troughs
fitted with electrodes.
3)Electric field of about 20 volts/cm is established.
4)The charged particles of sample migrate along the strip
towards respective electrodes of opposite polarity,
according to net charges, sizes and interactions with the
solid matrix.
5)Homogeneous group of particles migrate as a
separate band
6)The electrophoresis is carried out for 16-18 hours.
7)Proteins are stained (bromophenol blue) to make
them visible
8) The separated proteins appear as distinct bands.
FACTORS AFFECTING SEPARATION
1. The Sample-
• Charge- Higher the charge greater the mobility

• Size- Bigger the molecule greater the frictional and

electrostatic forces exerted on it by the medium i.e. larger
particles have smaller electrophoretic mobility
compared to smaller particles.
• Shape- The globular protein will migrate faster than the
fibrous protein MORE mobility
More mobility

2+
+
- 3+
16
2. Electric field-
Increase of migration with the increase of voltage
gradient.
MORE MIGRATION
20 v
+ - + - 10 v

3. Buffer- Migration of charge particle depend on of the

buffer.

a) composition
Commonly used buffers are “Formate”, “Acetate”,
“Citrate”, “ Phosphate”, “EDTA”

The choice of buffer depends upon the type of sample

17
being electrophoresed.
b) pH: The extent of ionization depends on pH, especially in
organic compounds. The ionization increases with
increase in pH of an organic acids and its just reverse for
the organic bases therefore affecting its rate of migration..

3.TheMedium :
The inert medium can exert adsorption ,molecular
sieving effects & electro-osmosis – processes that affect the
electrophoretic rate.

 Adsorption:
It means retention of a component on the surface of
supporting medium. The rate and resolution of
the electrophoretic separation can be efficiently reduced
by adsorption.
18
b) Molecular sieving:
Media such as “Polyacrylamide”, “Agar”, “Starch” &
“Sephadex” have cross-linked structures giving rise to
pores within the gel beads.

4) Heat generation in electric fields

One of the practical problems encountered in
electrophoresis is generation of heat from resistance in
the electrophoretic medium. Heating not only changes
viscosity and density of the electrophoretic media, it also
damages equipment.

19
 APPLICATIONS:

1) Paper electrophoresis has emerged as a simple,

inexpensive, and accurate laboratory procedure for
various research and clinical studies.
2) Clinical applications of paper electrophoresis include study
of sickle cell disease, hemoglobin abnormalities, and
separation of blood clotting factors and serum plasma
proteins from blood sample.

20
4) It has also been used in separation and identification of
alkaloids.

5)PE can also be used for testing water samples, toxicity of

water, and other environmental components.

6)Drug-testing industry uses paper electrophoresis to

21
determine presence of illegal drugs crime suspects.