This document provides an overview of electrophoresis. It defines electrophoresis as the migration of electrically charged particles or ions in solutions due to an applied electric field. It describes the basic theory behind electrophoresis, including how electrophoretic mobility determines the speed at which ions or solutes move. It also outlines the key components of an electrophoresis apparatus and describes the basic procedure for performing electrophoresis, including sample preparation, gel preparation, running the electrophoresis, and visualizing the results. Finally, it discusses several applications of electrophoresis across various fields like forensics, molecular biology, genetics, microbiology, and biochemistry.

1. ELECTROPHORESIS
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2. CONTENTS
Introduction
Theory
Electrophoresis Apparatus
Principle Of Electrophoresis
Procedure For Electrophoresis
Types Of Electrophoresis
Applications Of Electrophoresis

3. INTRODUCTION
 In practical terms a positive electrode(anode)
and negative electrode(cathode) are placed in a
solution containing ions.
Then voltage is applied across the electrodes, so
solute ions of different charge for example,
anions (negative) and cations (positive) will
move through the solution towards the electrode
of opposite charge.

4. -+
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ELECTROPHORESIS
The migration of electrically charged particles or ions in
solutions due to an applied electric field.
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5. THEORY
 Electrohoresis is the movement of ions under the
influence of electrical field. So, separation in
electrophoresis relies on differences in the speed of
migration (migration velocity) of ions or solutes.
• Migration Velocity (ν) =μe E
Where, μe=elecrophoretic mobility E=electrical field
strength
 Electrophoretic mobility is a factor that determines
how fast ion or solute moves in a given medium.
• μe = q/ 6Лήr
• where, q=charge of ion ή=viscosity of solution
r=radius of ion

6. The instrumentation of capillary electrophoresis is very similar to
that of HPLC.
Power supply of E is equivalent to HPLC pump and capillary
equivalent to column.
COMPARISON OF ELECTROPHORETICAND
CHROMATOGRAPHIC TERMS
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7. ELECTROPHORESIS APPARATUS

8. Principle of Electrophoresis
• Electromigration :
– Ions migrating in electric field
Cations cathode (-ve)
Anions  anode (+ve)
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9. Structure and Properties of Protein
• PROTEINS - polymers of amino acids
• STRUCTURE of AminoAcids
– aa's have a carboxyl group (-COOH) & amino group (-NH2) and
are often ionized at physiological pH
• Proteins are amphoteric compounds and are therefore either positively
or negatively charged.
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10. Migration of proteins in electric field
negatively charged proteins move towards the
positive pole
directly proportional to the overall charge of
proteins
inversely proportional to protein size (molecular
weight)

11. Electropherogram
An Electropherogram is the
result of an electrophoresis
which gives the movement of
charged particles over time in a
gel, paper or another medium.
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12. PROCEDURE FOR
ELECTROPHORESIS

13. 12
Melting the agar…

14. 13
Pouring the gel…

15. Solidification of gel

16. Creation of lanes

17. Formation of lanes

18. Preparation of sample

19. Buffer solution added to the tank
This ensures that the electric current goes through the whole tank and that maintains
that ions can move in the solution

20. samples loaded into wells

21. Electrical current applied to the chamber
Safety cover is put over the top and the current is switched on.
The dye will migrate through the gel toward the positive electrode, as will the DNA
Depending on how much voltage is applied and how warm the gel is and size and shape of
molecules will depend on how fast the ions move through the gel
Smaller fragments will move easier so they will be closer to the positive electrode
Once the dye has moved through the gel to the buffer, the electrical current is switched off
and gel is removed from the tray

22. VISUALISATION
• After the electrophoresis is complete, the molecules in the
gel can be stained to make them visible.
• Ethidium bromide, silver, or coomassie blue dye may
be used for this process.
• If the analyte molecules fluoresce under ultraviolet light,
a photograph can be taken of the gel under ultraviolet
lighting conditions.
• If the molecules to be separated contain radioactivity
added for visibility, an autoradiogram can be recorded of
the gel.

23. inversely proportional to the
logarithm
molecule.
of the size of the
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There are molecular weight size
markers available that contain a
mixture of molecules of known
sizes. If such a marker was run on
one lane in the gel parallel to the
unknown samples, the bands
observed can be compared to
those of the unknown in order to
determine their size. The distance
a band travels is approximately

24. Quantification of separated protein band by Densitometer
Densitometer is a device that measures
the degree of darkness in photographic
or semi-transparent material.

25. Types of Electrophoresis:
Zone Electrophoresis:
a) Paper electrophoresis
b) Gel electrophoresis
c) Cellulose acetate electrophoresis
d) Thin layer electrophoresis
 Moving boundary electrophoresis:
a) Capillary electrophoresis
b) Isoelectric focussing
c) Immuno electrophoresis

26. Applications of Electrophoresis
1. Forensics
DNA fingerprint of a criminal.
1. Molecular Biology To separate and organize DNA and RNA by
size
2. Genetics Provide clearer picture of DNA, it also helps prepare
DNA for cloning and genetic engineering.
3. Microbiology Information out about the organisms. Virology : to
help diagnose different strains of viruses.
4. Biochemistry Mapping of cellular components, particularly
proteins and nucleic acids.
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27. 5. Protein and peptide determination:
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(1) To check Purity
(2) Physical properties like MW, pI
(3) Binding studies
(4) Identification (CE-MS)
(5) Quantitation
(6) Immunoassays
1. Determination of additives in food and drug.
For example, Separation of Tartazine, Sunset yellow, amaranth,
indigo carmine
1.Separation and Quantification of Vitamins in fruits and
vegetable.
For example, Separation and quantification of Ascorbic acid in
vegetable and fruits
8. Used for separation of enatiomer from racemic mixture.

28. 9. Determination of minerals, fatty acids and carbohydrates.
1.Determination of various organic acids like Hippuric acid,
Oxalic acid, Tartaric acid, Malic acid, Lactic acid in food.
2. Pharmaceutical assay
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29. Thank you