This document discusses electrophoresis, which is a technique where charged molecules like proteins and nucleic acids migrate in an electrical field. The rate of migration depends on factors like the molecule's charge, size, shape, and the buffer properties. Electrophoresis is usually done with gels placed between two buffer chambers with electrodes. The key factors affecting electrophoresis are the sample properties, electric field strength, buffer composition and properties, and the supporting medium. Electrophoresis has applications in diagnostic testing and analyzing biological molecules like proteins.

1. Electrophoresis
principle and instrumentation, Factor affecting
Separation & it’s application
BY :-
AJAY PATIL
1st year m.pharm
( PHARMACEUTIC)
ABMRCP
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2. Electrophoresis
• Electrophoresis is a method whereby charged molecules in solution,
chiefly proteins and nucleic acids, migrate in response to an electrical
field.
• Their rate of migration through the electrical field, depends on the
strength of the field, on the net charge, size, and shape of the molecules,
and also on the ionic strength, viscosity, and temperature of the medium
in which the molecules are moving.
• As an analytical tool, electrophoresis is simple, rapid and highly sensitive.
• It can be used analytically to study the properties of a single charged
species or mixtures of molecules. It can also be used preparatively as a
separating technique
• Electrophoresis is usually done with gels formed in tubes, slabs,
or on a flat bed.
• In many electrophoresis units, the gel is mounted between two
buffer chambers containing separate electrodes, so that the
only electrical connection between the two chambers is through
the gel.

3. In most electrophoresis units, the gel is mounted between two buffer chambers containing
separate electrodes so that the only electrical connection between the two chambers is
through the gel.

4. The Technique

5. The Technique

6. Tube Gel Units

7. Slab Gel Units

8. Slab Gel Unit

9. Slab Gel Unit

10. Flat Bed Unit

11. FACTORS AFFECTING ELECTROPHORESIS
Electrophoresis is defined as the migration of charged particles through a
solution under influence of an electric field. Biological molecules such as
amino acids, peptides, protein, nucleotides, and nucleic acids possess
ionisable groups and aremade to exist as electrically charged species
either as cations or anions. Even carbohydrates can be given weak
charges by derivatization such as borates or phosphates. In
electrophoresis cation move towards cathode and anion move towards
anode.
The rate of migration is depends on
§ The charge of the particleApplied electric field
§ Temperature
§ Nature of the suspended medium

13. 1.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.
2. 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.

14. 3. BUFFER
Buffer determines & stabilizes pH of the supporting medium Also affects the migration rate of compounds in a number
of ways.
Composition of Buffer
§ Acetate
§ Barbiturate Citrate
§ EDTAFormate
§ Phosphate
§ Pyridine buffers commonly used.
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.
Low ionic strength
• Also decrease in overall current and hence decrease in heat production

15. 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.
4. 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.

16. Electro – endo-osmosis
Electro – endo-osmosis due to the presence of charged groups on the surface of the supporting medium
Eg. Paper - Carboxyl group (COO-)
Agarose - Sulphate group (SO2-)
Glass wall- Silanol (SiO-)
§ Above the pH value of three these charged groups will have ionize and generates negatively
charged sites. These ionized groups create an electrical double layer or region at supporting
medium.
§ When voltage is applied, cation in electrolyte near supporting medium migrate towards cathode
pulling electrolyte solution with them. This creates a net Electro – endo-osmotic flow towards the
cathode.
§ The Electro – endo-osmosis will accelerate the movement of cations, but retard anion
movements.

17. MOLECULAR SIEVING
§ Gels have sieve like structure.
§ In agar, starch, and poly acryl amide gels the movement of large molecule is hindered by
decreasing the pore size, since all the molecule has to transverse through pores.
§ If sephadex gel is used, small molecules are tightly held by pores and large molecules are
excluded by small pores causing movement outside the pores called molecular sieving.

18. APPLICATIONS
• Serum analysis for diagnostic purpose is routinely carried about by
paper electrophoresis.
• Muscle proteins, egg white proteins, milk proteins & snake, insect
venom analysis done by this technique.
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19. QUESTIONS

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