PAPER ELECTROPHORESIS.

In this slide contains types, working principle, factors affecting, advantage and disadvantage of paper electrophoresis.
Presented by: G.Sai Swetha. (Department of pharmacology),
RIPER, anantapur.

1. 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. 2 Introduction Paper electrophoresis Practical aspects Types of paper electrophoresis Working & Detection Factors affecting electrophoresis Advantages Disadvantages Applications Reference Contents

3. 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. 4 Electrophoresis

5. 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. 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. 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. 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. 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. 10 Horizontal paper electrophoresis

11. 11 Vertical paper electrophoresis

12. 12 Horizontal paper electrophoresis system Vertical paper electrophoresis system Paper electrophoresis system

13. 13 Vertical paper electrophoresis system Paper electrophoresis system

14. 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. 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. 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. 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. 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. 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. 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. 21 Serum analysis of proteins

22. 22 Paper Electrophorogram

23. 23 V . N. Kandula & S. Gummadi . A review on electrophoresis, capillary electrophoresis & hyphenations. IJPSR. 2020; 11(12): 6038-6056. W. P. Jencks. Paper electrophoresis as a quantitative method of serum proteins. NCBI. 1955; 60(2): 205- 215. Martin N. H. The use and limitations of filter paper electrophoresis. Journal of Clinical Pathology. 1952; 7(2): 87- 105. Myer Lubran . Paper electrophoresis. JAMA. 1996; 197(5): 360- 361. J. Sternberg. Paper electrophoresis. NCBI. 1953; 68(3): 284- 285. References

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