The document presents a seminar on paper electrophoresis as part of an M.Pharm curriculum, exploring its principles, types, practical aspects, and applications in separating biological molecules like proteins and amino acids. It discusses the working mechanism, required equipment, and the identification methods for compounds post-separation. Lastly, it highlights the advantages and disadvantages of the technique, along with its varied applications in serum analysis and environmental testing.

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A seminar as a part of curricular requirement
for I year M. Pharm. I semester.
Presented by
G. Sai Swetha, (20L81S0103)
M. Pharmacy
Department of Pharmacology
Under the guidance of
Dr. P. Ramalingam, M.Pharm., Ph. D
Research Director & Professor of Pharmaceutical and Medicinal Chemistry
Paper Electrophoresis
03-06-2021

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
1. Introduction
2. Paper electrophoresis
3. Practical aspects
4. Types of paper electrophoresis
5. Working & Detection
6. Factors affecting electrophoresis
7. Advantages
8. Disadvantages
9. Applications
10. Reference
Contents

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
• Electrophoresis refers to the migration of charged particles of
any size in liquid medium, under the influence of an electric
field.
• Depending on the kind of charge the molecule carry, they move
either towards anode or cathode.
• An ampholyte becomes positively charged in acidic condition
and migrates to cathode and in alkaline medium, they become
negatively charged and move to anode.
Introduction

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
Electrophoresis

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
1. Free electrophoresis
• Capillary electrophoresis
• Isotachophoresis
• Isoelectric focusing
• Immuno electrophoresis
2. Zone electrophoresis
• Paper electrophoresis
• Cellulose acetate electrophoresis
• Gel electrophoresis
• Thin layer electrophoresis
Types of electrophoresis

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
• Paper electrophoresis (PE) is a separation technique
• Mainly used to separate amino acids and proteins, peptides.
• Separation of mixture is based on charge and size of the
molecule.
• Migration of charged particles takes place through paper under
the influence of electric field.
• The paper strip used must contain at least α-cellulose at a percent
of 95% and should have a slight adsorption capacity.
• Paper does not contain charges that interfere with the separation
of compounds.
Paper electrophoresis

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
The equipment required include:
• A power pack- provides a stabilised DC and has controls for both
voltage output (0-500V) and current output (0- 150 mA).
• An electrophoretic cell- contains the electrodes, buffer reservoir, a
support for paper and a transparent insulating cover.
• Whatman filter paper, cellulose acetate filter paper or
chromatography paper are used in PE.
• Prior to use, the paper is washed with distilled water followed by
0.1M HCl or 0.01M EDTA to remove impurities.
Practical aspects of PE

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Based on voltage applied, it is of two types:
• Low voltage PE
- a voltage of 100-300 V is applied across the electrodes
- more no. of samples can be separated on a single paper, at a time
- diffused sample bands may appear
• High voltage PE
- up to 1000 V is applied across the electrodes
- sharp bands are obtained
- more voltage is required so, it is dangerous to operate
Types of paper electrophoresis

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
Based on design of instrument, it is of two types:
1. Horizontal paper electrophoresis
2. Vertical paper electrophoresis
The two arrangements of the filter strips are commonly used. Both
the arrangements are equally viable and the choice usually
depends upon personal preferences.
Types of paper electrophoresis

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
Horizontal paper electrophoresis

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
Vertical paper electrophoresis

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
Horizontal paper electrophoresis system Vertical paper electrophoresis system
Paper electrophoresis system

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Vertical paper electrophoresis system
Paper electrophoresis system

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
• 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.
Working

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
Some commonly used buffers include:
Intended
Separation
Buffer pH
Proteins Barbital 8.6
Phosphate 7.4
Nucleoproteins Acetate 4.5
Citrate 4.5
Amino acids Phosphate 4.6
Michaelis 8.6

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
Individual compounds are identified by 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.
Detection

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Compound Dye
Proteins Bromophenol blue in Acetic acid,
DANSYl Chloride
Nucleic acids Methyl-green pyronine,
Ethidium bromide
Polysaccharides Iodine
Glycoproteins Alcian blue
3. Staining

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
Factors affecting electrophoresis:
• Properties of 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

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
19
Advantages
• It is simple and inexpensive process.
• It is easily available and easy to handle.
Disadvantages
• Certain compounds like proteins, hydrophilic molecules cannot be
resolved because of adsorptive and property of paper.
• It results in tailing and distortion of component bands.
• Electro osmotic flow may occur.
• Time taking process. It takes almost 14-16 hours for separation
process.

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20
• 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.
Applications

21. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 21
Serum analysis of proteins

22. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 22
Paper Electrophorogram

23. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 23
1. V. N. Kandula & S. Gummadi. A review on electrophoresis,
capillary electrophoresis & hyphenations. IJPSR. 2020; 11(12):
6038-6056.
2. W. P. Jencks. Paper electrophoresis as a quantitative method of
serum proteins. NCBI. 1955; 60(2): 205- 215.
3. Martin N. H. The use and limitations of filter paper electrophoresis.
Journal of Clinical Pathology. 1952; 7(2): 87- 105.
4. Myer Lubran. Paper electrophoresis. JAMA. 1996; 197(5): 360-
361.
5. J. Sternberg. Paper electrophoresis. NCBI. 1953; 68(3): 284- 285.
References

24. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 24