Paper electrophoresis is a technique used to separate charged molecules like amino acids, peptides, and proteins. It works by applying an electric current across a paper strip soaked in buffer solution, which causes the charged molecules in a sample to migrate across the paper at different rates based on their charge and size. The document discusses the principle, requirements, procedure, advantages, types (based on voltage and design), and factors affecting migration of molecules in paper electrophoresis. It is a simple, inexpensive, and widely-used method to qualitatively analyze biological samples for clinical diagnosis and research purposes.

1. PAPER
ELECTROPHORESIS
R Deepthi
Assistant Professor
Vignan Institute of Pharmaceutical Technology
Visakhapatnam

2. Paper Electrophoresis
Paper electrophoresis is the most extensively used technique for separation
of substances like amino acids, peptides and proteins.
Electrophoretic analysis (using paper) of plasma proteins is of clinical
significance in the diagnosis of various diseases including multiple
myeloma, cirrhosis and nephrosis.
Advantages
This technique is very simple and inexpensive.
Low operational costs.
Numerous samples can be simultaneously isolated on a single paper.
Various substances including amino acids, proteins, peptides, antibiotics,
alkaloids etc. can be easily isolated by this technique.

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 (m/e)
ratios.
As paper does not conduct electricity, it is wetted with a buffer for
facilitating the transport of current.
The particles or ions present in the sample move either towards the
cathode or the anode depending on the charge they possess.

6. Migration of ions also depends on the following factors:
1. Charge of the Particle: Rate of migration of the ions is directly proportional to the
charge on the molecule. Ex: X2+ ions migrate faster when compared to X+ ions.
2. Size and shape of the molecule: Rate of migration of ions is inversely proportional to
their size, hence smaller ions migrate faster when compared to larger ions.
According to Stoke’s law, electrophoretic mobility of ion is expressed as,
µ = Q/6πrɳ
Where,
µ = Electrophoretic mobility of the ion
Q = Charge of the ion
R = Radius of the ion
ɳ = Viscosity of the buffer solution

7. 3. Viscosity of the Buffer solution: Electrophoretic mobility of ion is
inversely proportional to viscosity of the buffer solution i.e., rate of
migration of ion decreases with an increase in the viscosity of buffer
solution.
4. Voltage applied: Rate of migration of ion is directly proportional to the
voltage applied across the electrodes. Therefore, mobility of ions can be
enhanced by increasing the voltage.
Furthermore, application of high voltage produces sharp bands on paper
which are easy to detect. However, care should be taken to minimize the
evaporation of buffer solution due to high voltage.

8. 5. pH and ionic Strength of the Buffer solution: Rate of migration of ions
is inversely proportional to the ionic strength of the buffer solution i.e.,
ionic mobility increases with decrease in ionic strength.
Influence of pH on rate of migration of ions depends on whether pH of
the buffer solution is above or below the isoelectric point of the sample
to be analyzed.

10. Requirements of Paper Electrophoresis
1. Paper
In paper electrophoresis, various types of papers are used as stabilizing agents
including Whatmann 1,2,3; Eaton-Dikemann 301-85, 320, 352, Munketells
20/50, Schleicher and Schull 2040 A and B etc.
Of all those mentioned above, Whatmann 1 is extensively used particularly for
large samples greater than 20µl.
For samples which react with paper, paper made up of borosilicate glass fibres
like whatmann GF/B are used. In general, filter paper obtained from various
manufacturers differ in terms of thickness, optical homogeneity and content of
foreign materials.
Hence, filter papers are 1st washed with distilled water and then with 0.1M HCl
or 0.01M EDTA to remove any impurities.

11. 2. Electrodes
Graphite rods, stainless steel, silver chloride, platinum (Pt) are the most
commonly used electrode materials in paper electrophoresis.
Platinum electrode is most extensively used and is available in various
forms such as fine wires, sheets of foil etc.
 Size of the electrodes should not be small as they possess greater risk of
being polarized with gas bubbles or products of electrolysis and also limit
the amount of current flow.

12. 3. Buffers
In general, ionic strength of about 0.05-0.5M is commonly used in paper
electrophoresis.
Examples of buffering agents are 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 0.07mole/Lt and pH 7.6 and citrate buffer at a
concentration of 0.07moles/Lt and pH 3.0 or 6.8.

13. Procedure
In paper electrophoresis, the stabilizing medium i.e., filter paper is 1st
dipped into the buffer solution and excess buffer is removed by placing it
on another sheet of paper.
Prior wetting of the filter paper is essential as it ensures uniform
distribution of the buffer.
After removing excess buffer solution, the paper is laid across two
beakers containing the buffer solution of known ionic strength and the
ends of the paper strip are immersed into the beakers

14.  Paper is then allowed to stand for some time and the apparatus is enclosed
in an air tight chamber so as to prevent excessive loss of buffer solution
due to evaporation.
Sample is then introduced on paper as a band/spot at its centre with proper
care. Voltage potential is then applied across the electrodes immersed in
the beakers.
Care should be taken to ensure that paper and electrodes are sufficiently
isolated so as to prevent electrode reactions which occur due to changes in
the composition of buffer solution.

15. When voltage is applied, the ions and ionizable substances present in the
sample to be analyzed migrate towards their respective electrodes based
on their charge.
 The spots/bands obtained on filter paper are detected using suitable
visualizing agents as in paper chromatography.
This technique is extensively used for both Qualitative and Quantitative
analysis of the sample.

16. Types of Paper Electrophoresis
1. Based on Voltage Potential:
Depending upon the potential applied across the electrodes, paper
electrophoresis is broadly classified into two types:
i) Low voltage paper Electrophoresis: In this type, voltage applied across
the two electrodes ranges from 5-15V/cm or 100-300V/strip with a
current of about 0.4mApm/cm or 1.5mAmp/strip.
 Low voltage paper electrophoresis is widely used for the separation of
ions for laboratory purpose.

17. ii) High voltage Paper Electrophoresis: In this type, voltage applied across
the two electrodes ranges from 50-215 V/cm or 10,000V/strip.
 Here, isolation of ions requires less time and sharp bands are obtained.
 Hence, similar compounds can be easily separated and more number of
samples can be analyzed at the same time.
However, it is very hazardous thus requiring proper care. Furthermore, a
large amount of heat is produced due to high voltage as a result of which
the paper may become dry when the buffer evaporates.

18. 2. Based on Design of the Instrument:
Based on the design of the instrument, paper electrophoresis is broadly
classified into three types. The principle involved in the operation of all
the three types is same but the design of the instrument varies.
i) Horizontal Type Paper Electrophoresis: In this type, Whatmann filter
paper of suitable grade and dimensions is moistened with buffer solution
of known ionic strength and pH.
Excess buffer solution is removed and the sample is applied at the centre of
the paper.

19. The paper is then supported horizontally between two glass or plastic
plates so as to prevent the evaporation of buffer solution.
 When suitable potential is applied across electrodes, migration of ions to
their respective electrodes takes place.
 Spots or bands obtained on the filter paper due to migration of ions are
detected by using a suitable visualizing agent. In horizontal type,
separation of ions takes place within 12-14 hrs.

21. Vertical Type Paper Electrophoresis: It is similar to horizontal type
except that here the paper is supported at some angle between glass or
plates.
In this the rate of migration of ions depends upon gravity hence complete
separation of ions takes place within 6-8hrs.
Quantitative detection of bands/spots is carried out by densitometer.

23. iii) Continuous Paper Electrophoresis: In this type, buffer solution is made
to flow over the paper bed at a constant rate while the sample stream
flows across the bed in the same direction as that of buffer.
When potential is applied, migration of ions to their respective electrodes
takes place.
Isolation of ions takes place vertically, owing to the differences in
distribution ratio between the mobile and stationary phase.
Thus, each band is made to fall down and pure compounds obtained are
collected in separate beakers as shown in the figure below:

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