This document discusses different electrophoresis techniques used to separate biomolecules like proteins, nucleic acids, and lipids. It describes gel electrophoresis where molecules are separated in a gel matrix under an applied electric field based on size and charge. Key aspects covered include the instrumentation, supporting media like agarose and polyacrylamide gels, detection methods using stains, and applications like DNA sequencing and protein purification. Moving boundary electrophoresis is also summarized, which allows separation in solution without a supporting medium by formation of distinct boundaries between charged species.

1. Submitted By:- Subham Kumar Vishwakarma
M.Pharm-I
Pharmaceutical Chemistry
GITAM Institute of Pharmacy Visakhapatnam

2. ELECTROPHORESIS
• The word electrophoresis is derived from a Greek word, which means
borne by electricity.
• It is a separation technique in which the components are separated due to
their varying behavior under the influence of applied electric field.
• It is defined as the migration of charged molecules under the influence of
external electric field.
• The major requirement of the component to be subjected to
electrophoresis is that the component should be charged.

3. ELECTROPHORESIS
• The relative mobility of individual molecule depends on several factors.
The most important of which are:
1. Charge- higher the charge greater the electrophoresis mobility.
2. Size - bigger the molecule, greater are the frictional and electrostatic
forces exerted on it by the medium. Consequently larger particle
have smaller electrophoretic mobility compared to smaller particles.
3. Molecular shape – rounded counters elicit lesser frictional and
electrostatic retardation compared to sharp counters. Therefore,
globular protein move faster than fibrous ones.

4. GEL ELECTROPHORESIS
• Gel electrophoresis is a method for separation and analysis of
macromolecules like DNA, RNAand proteins or their fragments, based
on their size and charge.
• Gel electrophoresis uses a gel as an anti-convective medium and/or
sieving medium during electrophoresis.
• Gels suppress the thermal convection caused by application of the
electric field, gels can also simply serve to maintain the finished
separation, so that a post electrophoresis stain can beapplied.
It is carried out by using
• Agar
• Starch
• Polyacrylamide

5. Principle
• By placing the substance to be separated in
wells of the gel and applying an electric
current, allows the molecule to move
through the matrix at different rates
towards the anode if negatively charged or
toward the cathode if positively charged.
• As they move through the gel, the larger molecules will be held up as
they try to pass through the pores of the gel, while the smaller molecules
will be impeded less and move faster.
• This results in a separation by size, with the larger molecules nearer the
well and the smaller molecules farther away.

6. Principle of separation
• According to charge: When charged molecules are placed in an electric
field, they migrate toward either the positive (anode) or negative
(cathode) pole according to their charge.
• According to size: The smaller molecules move more swiftly than the
larger sized ones, as they can travel through the pores more easily than the
later.

7. Instrumentation
1. Electrophoresis apparatus
2. Buffer
3. Power supply
4. Supporting media
5. Detection and Quantification

8. 1) Electrophoresis apparatus:
• The casting tray is made up of glass
or plastic.
• The comb contains varying number
of teeth in order to help in formation
of well.
o Electrophoresis apparatus set up:
Electrophoresis chamber with buffer solution
Casting tray
Electrodes

9. 2) Buffer
• Buffers in gel electrophoresis
are used to provide ions that
carry a current and to maintain
the pH at a relatively constant
value.
• The most common being, for
nucleic acids
Tris/Acetate/EDTA (TAE),
Tris/Borate/EDTA(TBE).

10. 3) Power Supply
• The electrodes are connected to
their respective terminals of the
electrophoresis chamber and to the
power supplier with controls for
rate of current flow.
• The best resolution of fragments
larger than about 2 kb is attained by
applying not more than 5 volts per cm
to the gel.

11. 4) Supporting media: (Gel)
1. Starch
2. Agar/agarose
3. Cellulose acetate
4. Polyacrylamide gel
• The kind of supporting matrix used depends on type of molecules to
be separated and the desired basis for separation: charge, molecular
weight or both
• Agarose and polyacrylamide gels are cross-linked, spongelike
structure

12. Cont.
• It is important that the support media is electrically neutral. Presence
of charge group may cause:
o Migration retardation
o The flow of water toward one or the other electrode so called
“Electroendosmosis (EEO)”, which decrease resolution of the
separation
• Agarose Gels have fairly large pore sizes and are used for separating
larger DNA molecules (Restriction Fragment Length Polymorphism
Analysis)
• Polyacrylamide Gels are used to obtain high resolution separations
for smaller DNA molecules (STR analysis-Short Tandem Repeat
analysis and DNA sequence analysis)

13. 5) Detection and quantification:
• Stains
o Protein staining
o Ethidium bromide staining
• Blotting
o Southern blotting (for DNA)
o Northern blotting (for RNA)
o Western blotting (for protein)

14. Ethidium
bromide
Protein staining

15. Process of gel electrophoresis

16. Applications
• Separation of Deoxyribonucleic acid
• Separation of ribonucleic acid
• Separation of protein molecules
• It may be used as preparative technique prior to use of other
methods such as mass spectroscopy, cloning, DNA Sequences,
Southern Blotting for further characterization.
• Separation of amino acid
• Separation of lipoproteins
• Separation of enzyme in blood
• Separation of antibiotic drug

17. MOVING BOUNDARY ELECTROPHORESIS
Principle:- It allows charged species to migrate in a free moving
solution, without the supporting medium
The main features of this method are:
• The formation of sharp boundaries
• Large electrode vessels containing reversible electrodes
• An optical system for following movement of boundaries
• Thermostatic control

18. INSTRUMENTATION
• Apparatus consists of U tube, with electrodes located at the two ends
used to apply an electric field.
• The lower part of the cell is filled with lyophillic solution under
examination, sometimes the sample solution is introduced into the
bottom of the U tube through a capillary arm, while upper part
contains only the buffer solution.
• Commonly used buffers are- Formate, Acetate, Citrate, Phosphate
• Care must be taken to minimize the disturbing effect of convection
caused by an increase in temperature during the passage of current
through the solution.
• For this purpose, the apparatus is placed in constant temperature bath
at 4°c.

21. APPLICATIONS
• To study the homogenecity of a macromolecular system.
• It is employed in biochemical and clinical fields i.e in the study of
protei mixtures such as blood serum, haemoglobins and in the study of
antigen-antibody interactions.
• It is also used for the separation of scopolamine and ephedrine using
buffer with pH 4.2
• Protein research/purification.

22. REFERENCE
Instrumental methods of chemical analysis By Dr.
B.K.Sharma, Page no. 661-670.
Dely z., Electrophoresis , Elsevier scientific publishing company,
p.no.156-158
Robinson J. W., Undergraduate instrumental analysis, 6th edition, 2009,
Marce