This document discusses electrophoresis, which is a technique used to separate ions or molecules based on their size and charge. When an electric field is applied, positively charged ions migrate toward the negative electrode and negatively charged ions migrate toward the positive electrode. Electrophoresis can be used to determine components in a sample, obtain information about electrical double layers, and determine molecular weights of proteins and DNA sequencing. Factors like the nature of the analyte, electric field properties, buffer used, and temperature affect electrophoretic separation. Different types of electrophoresis are described including free solution, moving boundary, zone, gel, paper, cellulose acetate, and continuous electrophoresis.

1. ELECTROPHORESIS
BINDU KSHTRIYA

2. ELECTROPHORESIS
• Electrophoresis is a technique in which ions in solution are
separated based on their differences in size and charge.
• When a high voltage is applied to the solution positive ion
migrates toward the negative electrode and negative ions
migrates to the positive electrode.

3. GENERAL PURPOSE OF ELECTROPHORESIS
• Determine the number, amount and mobility of a
components in a given sample or to separate them.
• To obtain information about the electrical double
layers surrounding the particles.
• Determination of molecular weight of proteins and DNA
sequencing.

4. PRINCIPLE
• Any charged ion or molecule migrates when placed in an electric field.
The rate of migration depend upon its net charge, size, shape and the
applied electric current.

6. ELECTROPHORETIC MOBILITY
• The movement of charged particle in an
electric field is expressed in terms of
electrophoretic mobility denoted by µ.
where
µ = v/E or µ = q/f

7. FACTORS AFFECTING ELECTROPHORETIC
SEPARATION:
A. ANALYTE- nature of the compound affects the
separation in many ways-
1. CHARGE– higher the charge, greater is the electrophoretic
mobility.
2. SIZE-bigger the molecule greater are the frictional and
electrostatic forces exerted on it by the medium. Consequently,
larger particles have smaller electrophoretic mobility compared
to smaller particles
3. SHAPE- rounded molecules elicit lesser friction compared to
sharper molecules.
e.g. globular protein move faster than fibrous protein.

8. B.ELECTRIC FIELD- according to Ohm’s law the current
flowing through the solution is given by
I=V/R
where I= current
V= voltage
R= resistance
1. VOLTAGE: regulates the current , rate of migration is
proportional to voltage applied.
2. CURRENT: rate of migration is directly proportional to
the current.
3. RESISTANCE: rate of migration is inversely
proportional to resistance.

9. C.BUFFER- the nature of buffer used as an electrolyte is
extremely important and it influences the success of the
separation.
pH
The product of net charge & the field strength provides the
total motive power for electrophoretic separation.
Since the net charge carried by each species in the solution
is pH dependent, therefore, rate of migration of ion is
affected by the pH of the medium.
D.TEMPERATURE- the mobility of ions increases with increase
in temperature upto certain level, but higher level of
temperature causes evaporation of solvent from bed.

10. TYPES OF ELECTROPHORESIS

11. FREE SOLUTION
ELECTROPHORESIS
• In this, the sample solution is introduced as a
bond at the bottom of U tube that has been filled
with unstabilised buffer solution.
• The sample are usually injected into the bottom
of the U tube through a capillary tube side arm.
• An electric field is applied and separation takes
place as result of differences in mobilities.

12. MOVING BOUNDARY
ELECTROPHORESIS
• U shaped tube is filled with buffer.
• Sample is injected at the bottom of the U-
tube through a capillary
• An electrical field is then applied.
• Separation takes place as a result of
differences in mobilities.
• Different fractions obtained are located by
optical measurement using optical system
i.e. by measuring difference in Refractive
indices.

14. ADVANTAGES-
• Greater sensitivity.
• Better separation.
• Direct method to separate solute from buffer solution.
• Concentration as low as 0.05 mg/ml can be detected.
DISADVANTAGES-
• High equipment cost .
• Optical system required.
APPLICATIONS-
• For analytical purpose –Protein mixture.
• To study colliding dispersion.

15. ZONE ELECTROPHORESIS
The separation is carried out in a stabilizing medium or
supporting medium.
Types of supporting medium used :
• Filter Paper
• Cellulose Acetate
• Starch powder
• Starch gel
• Cellulose powder
• Agar gel
• Agarose gel

16. PAPER ELECTROPHORESIS
It is the form of electrophoresis that is carried
out on filter paper. This technique is useful for
separation of small charged molecules such
as amino acids and small proteins.
FILTER PAPER : It is the stabilizing medium.
We can use what-man filter paper, cellulose
acetate filter paper or chromatography paper.
APPARATUS : Power pack, electrophoretic
cell that contains electrodes, buffer
reservoirs, support for paper, transparent
insulating cover

17. SAMPLE INJECTION
The sample may be applied as a spot(about 0.5 cm in diameter)or
as a uniform streak.
ELECTROPHORETIC RUN :
The current is switched on after the sample has been applied to
the paper and the paper has been equilibrated with the buffer. The
types of buffer used depends upon the type of separation. Once
removed, the paper is dried in vacuum oven.
DETECTION AND QUANTITATIVE ASSAY:
Fluorescence, ultraviolet absorption or radioactivity are
exploited for detection.

18. CELLULOSE ACETATE ELECTROPHORESIS
Cellulose acetate is more homogeneous and stabilizes fluid more
efficiently, it gives better resolution than the paper.
Other advantages:
➢ Free of surfaces charges
➢ Can be made transparent for densitometry by treating
it with plasticizing agent
➢ Smaller sample capacity than paper

19. Strip of electrophoresis
Electrophoresis
Major protein components
separate into discrete
zones
Densitometer tracing density of
zone to the amount of protein

20. GEL ELECTROPHORESIS
Involves the use of a
gelatinous material such as
Agarose, polyacrylamide,
starch as the matrix.
The gel acts as a support
medium for the sample.

21. AGAROSE
ELECTROPHORESIS
▪A highly purified uncharged polysaccharide
derived from agar.
▪Used to separate macromolecules such as
nucleic acids, large proteins and protein complexes.
▪It is prepared by dissolving 0.5% Agarose in
boiling water and allowing it to cool to 40°C.
▪It is fragile because of the formation of weak
hydrogen bonds and hydrophobic bonds

22. POLYACRYLAMIDE GEL
▪ Components: Acrylamide monomers, Ammonium
persulphate, Tetramethylenediamine (TEMED)
Etc.
▪These free radicals activate acrylamide monomers
inducing them to react with other acrylamide
monomers forming long chains.

23. SDS-POLYACRYLAMIDE GEL
ELECTROPHORESIS
• SDS (also called lauryl sulfate) - anionic detergent
• Molecules in solution with SDS have a net negative
charge within a wide pH range.
• A polypeptide chain binds amounts of SDS in
proportion to its relative molecular mass.
• The negative charges on SDS destroy most of the
complex structure of proteins, and are strongly
attracted toward an anode (positively-charged
electrode) in an electric field.

24. ADVANTAGES OF ZONE
ELECTROPHORESIS
▪ SIMPLE AND INEXPENSIVE.
▪ HIGH RESOLUTION POWER.
▪ SMALL QUANTITY OF SAMPLE CAN BE ANALYSED.
▪ DETERMINATION OF LOW MOLECULAR WEIGHT SUBSTANCES.

25. ZONE ELECTROPHORESIS
A D V A N T A G E S :
▪ Simple and inexpensive.
▪ High resolution power.
▪ Small quantity of sample
can be analyzed.

26. ▪ DISADVANTAGES:
Less accurate.
Excessive spreading of zones.
Absorption of solute takes place.

27. APPLICATIONS OF ZONE
ELECTROPHORESIS
▪ In clinical ,diagnosis, analysis of serum, urine and
other body fluids.
▪ Convenient method of separating the inorganic ions.
▪ Desirable to measure the special kinds of proteins.
▪ For the study of chemical coagulation of water.

28. CONTINUOUS ELECTROPHORESIS
• A thin sheet of filter paper is usually used as a supporting
medium.
• Buffer solution is made to flow over the bed at a uniform
rate.
• Sample to be separated is applied in small stream that
• flows in the line across the bed in the same direction as
a buffer.
• Potential difference is applied across the electrodes, ions
• will migrate at different rates, and the components of the
• sample are carried out down by the flow of
buffer solution.
• Separation occurs at vertical direction.

29. APPLICATIONS OF CONTINUOUS
ELECTROPHORESIS
▪ Determination of variety of charged species.
▪ Determination of low molecular ions, membranes, and
cells.
▪ Optimization for the fast and efficient separation.

30. THANK YOU
BINDU KSHTRIYA