Principles, applications, types of electrophoresis.

2. Electrophoresis
• INTRODUCTION:
• Electrophoresis: It is a separation method based on
differential rate of migration of charged species in an applied
dc electric field.
• Rate of migration
• Depends on charge and size
• Separation based on differences in
charge-to-size ratios

3. Historical background
• Initially developed by Arne Tiselius in the 1930’s –
Separated serum proteins
• Slab gel electrophoresis: developed in the 1950’s
• Capillary electrophoresis: – developed in the 1980’s –
Narrow bore tubes used
• Applications of electrophoresis: – Separation of
proteins and nucleic acids (single nucleotide
differentiation capability!) – The human genome
Project
• • Human DNA: ~three billion nucleotides

4. Electrophoresisprinciple:
• Principle involves electrophoretic mobility of
ions.
• It has been found that the migration of
velocity(v) (cms-1) of molecule in an electric
field is given by
v = (µe E)
µ = electrophoretic mobility
E = strength of electric field

5. • The value of (E) depend upon:
• Charge of the analyte ion.
• The friction regarding force.
• Which includes
• Size and shape of ion.
• Viscosity of the medium in
which migration occur.

6. Instrumentation:
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.

7. The technique:

8. Tube gel unit

9. Slab gel unit

10. Slab gel unit

11. Flat bed unit

12. Type of electrophoresis
• Zone electrophoresis:
• Paper electrophoresis
• Gel Electrophoresis
• Thin layer Electrophoresis
• Cellulose acetate Electrophoresis
• Moving boundary electrophoresis:
• Capillary electrophoresis
• Isotachophoresis
• Isoelectric focusing
• Immuno Electrophoresis

13. Zone Electrophoresis
• It involves migration of charge particle on the supporting
media.
• Such as paper, cellulose acetate membrane, Starch gel,
poly acrylamide.
• supporting media always saturated with buffer.
• Advantages:
• Useful in biochemical investigation.
• Cost is low and easy maintenance.
• Disadvantages:
• Unsuitable for accurate mobility.

14. Moving boundary electrophoresis
• It contain U shaped arrangement.
• It does not need any supporting media to move.
• High mobility, fast migration.
• Low mobility , slow migration.
• Used for separation of protein as well as inorganic
substances.

15. Capillary electrophoresis:
• Capillary electrophoresis is a technique of performing
electrophoresis in buffer filled, narrow-bore capillaries
normally from 25-100 mm internal diameter.
• High voltage typically 10-30 kV.
•

16. Isotachophoresis
• This technique depend upon the development of potential
gradient.
• Based on principle of moving boundary electrophoresis.
•

17. Isoelectric focusing
• All proteins have an isoelectric point.
• This will be in zwitter ion form no electric charge so,
further moment will be cease.
• Protein migrate to the point where its net charge is 0, so,
isoelectric pH.
• Use for enzymology, immunology,
cytology and taxonomy.

18. Immuno Electrophoresis
• Antibodies are produce by immune system in response to
foreign macromolecules (antigen).
• When electric potential is applied to study antibody-
antigen reactions, it is called Immuno electrophoresis.
• Mainly used for separating proteins and poly peptides.
• Used in forensic , clinic and human genome laboratories
for separation.
• Used in identification of serum protein in research in
enzymology, membrane biochemistry, microbiology and
immunology.

19. Detectors used in electrophoresis
• UV absorption detector: used for uv absorbing
materials.
• Conductivity detector: used for measuring electric
resistance and conductivity.
• High frequency detector: Used for field strength.
• Thermometric detector: Used for temperature and heat
production. By measuring temperature of particular ionic
species location can be found out.

20. Application of elecrophoresis
• Gene and genome Analysis: Electrophoresis used for
analyzing gene. The analyzed DNA used in forensic
investigations.
• Protein and proteome analysis: Amount of protein in blood
and urine can be analyzed.
• Antibiotic Analysis: Electrophoresis used for new type of
antibiotics such as penicillin (drug used for bacterial
infection).
• Vaccine Analysis: electrophoresis used for purification,
processing and analyzing various vaccine.

21. By :
Scholar Hina Islam Chishty
Organic chemistry
Institute of Chemistry
E-mail: chishtyislam555@gmail.com
Shah Abdul latif university
Khairpur Mirs` Sindh
Pakistan