Paper Electrophoresis - Defenition, principle, types of Paper Electrophoresis, components, working procedure, application, advantages and disadvantages.

1. SUBJECT: BIOINSTRUMENTATION
TITLE : PAPER ELECTROPHORESIS
SEMINARASSIGNED DATE : 12.08.2025
SEMINARCOMPLETION DATE : 18.08.2025
SUBMITTED BY:
A.Daisy Beula
I M.Sc. Microbiology
Sri Paramakalyani College,
Alwarkuruchi.
SUBMITTED TO:
PG and Research Department of
Microbiology,
Sri Paramakalyani College,
Alwarkuruchi.

2. Paper Electrophoresis
• Paper electrophoresis is a separation technique.
• Mainly used to separate amino acids, proteins and peptides.
• Separation of mixture is based on charge and size of the molecule.
• Electrophoretic analysis (using paper) of plasma proteins is of clinical
significance in the diagnosis of various disease including multiple
myeloma, cirrhosis and nephrosis.

3. Principle
The principle involved in paper electrophoresis is the separation of charged
particles from the sample applied on a paper upon application of current
using two electrodes.
The rate of separation of particles is based on their mass to charge ratios.
Paper does not conduct electricity it is wetted with a buffer for facilitating
the transport of current.
The ions present in the sample move either towards the anode or cathode
depends on the charge they possess.

4. Components
Paper :
paper is used as the supporting medium. Whatman filter paper of suitable
dimensions with a length so that both end of the strip of the paper touches the
buffer solution and kept in the electrolyde vessels.
Electrodes and voltage to be applied:
The electrode in the form of a thin wire is made up of carbon or platinum, A
DC voltage of about 8- 15V/cm length of paper is normally applied.

5. Buffer
In general, ionic strength of about 0.05-0.5M is commonly used in paper
electorphoresis.
Examples of buffering agents used are barbitone buffer or veronal buffer at a
concentration of 0.07 moles/lt and pH 8.6,
tris-acetate buffer at a concentration of 0.07moles/lt and ph 7.6 and citrate
buffer at a concentration of 0.07moles/lt and pH 3.0 or 6.8.

6. Types of Electrophoresis
Based on voltage applied, they are two types
• Low voltage paper electrophoresis
A voltage of 100- 300v is applied across the electrodes.
More number of samples can be separated on a single paper at a time.
Diffused samples bands may appear
• High voltage paper electrophoresis
Up to 1000v is applied across the cathodes.
Sharp bands are appear.
More voltage is applied so it is dangerous to operate.

7. Based on design of the instrument:
Based on the design of the instrument, paper electrophoresis is
classifird into three types
i. Horizontal type paper electrphoresis
ii. Vertical type paper electrophoresis
iii. Continous paper electrophoresis

8. Horizontal type paper electrophresis
In horizontal paper electrophoresis buffer solution of known pH and ionic
strength is filled in two beakers. Whatman filter paper of suitable grade and
convenient dimensions are cut and immersed in buffer solution. Sample
solution is applied at the centre of the paper and fixed in position.

9. Vertical paper electrophoresis
• The migration of ions takes place by the gravity. After sufficient migration
the paper is taken out and dried to fix the spots.
• Then the spots can be visualised like in paper chromatography. Quantitation
of spots can also be carried out using densitometer.

10. • .

11. Continuous paper electrophoresis
• In this type predetermined sample volume through a value device is applied
continuously on the centre of the paper.
• Suitable voltage cause migration of samples and compounds separated as
bands. Thus each band is made to fall down and pure compounds are
collected in the separate containers.

12. Working
• A paper strip of suitable length is moistened with suitable buffer of required
pH and the sample is applied transversely across the central part of paper
strip.
• Ends are fixed to dip in buffer solution in two troughs, fitted with electrodes.
• Now electric field is applied across the system.
• The charged particles of sample migrate along the strip towards respective
electrodes of opposite polarity.
• Homogenous group of particles migrate as separate band.
• The process is carried out for 14-16 hours.

13. Some comnonly used buffers are:
Intended
separation
Buffer pH
Proteins Barbital 8.6
phosphate 7.4
Nucleproteins Acetate 4.5
Citrate 4.5
Amino Acids Phosphate 4.6
Michaelis 8.6

14. Detection
Individual components are identified by the following methods
1. UV absorption
Proteins, peptides and nucleic acids absorb UV radiation at a
range of 260 to 280 nm.
2. Fluorescence
Staining with Ethidium bromide and observing the
electrophorogram under UV light makes DNA and RNA flouresce and
fascilitates detection.
Flourescamine staining is used for detecting amino acids,
peptides, proteins.

15. 3. Staining
Compound Dye
Proteins Brpomophenol blue in
Acetic acid, Dansyl chloride
Nucleic Acid Methyl-green pyronine,
Ethidium bromide
Polysaccharides Iodine
Glycoproteins Alcian blue

16. FactorsAffecting Electrophoresis
• Properties for supporting medium
• The strength of electric field
• Temperature of the buffer
• pH of the buffer
• Molecular mass of the sample
• Net charge on the particles
• Shape and size of the particles

17. • It is simple and inexpensive process.
• It is easily available and easy to handle.
Disadvantages
• Certain compounds like protein, hydrophilic molecules cannot be resolved
because of property of paper
• It results in tailing and distortion of component bands.
• Electro osmotic flow may occurs
• Time taking process, it takes almost 14-16 hours for separation process.
Advantages

18. Application
• Serum analysis for diagnostic purpose is routinely carried out by paper
electrophoresis.
• Separation of amino acids, proteins in serum.
• Separation on enzymes in blood.
• Separation of antibiotics in different samples.
• Muscle proteins, egg white proteins, and snake, insect venom analysis is
done by this technique.
• Used in the separation and identification of alkaloids.
• It can also be used to test the suitability of municipal water supplies, the
toxicity of water, and other environmental components.

19. • An old owl watches the world from its tree, absorbing wisdom without
speaking much. As it observes the actions and words of others, it
learns important life lessons. This owl teaches us that listening and
observing can lead to greater knowledge than merely speaking.
• Moral: Talk less, observe more.