Application of paper electrophoresis

This presentation includes the main applications of the paper electrophoresis on the basis of the principle given

1. APPLICATION OF PAPER ELECTROPHORESIS SUBMITTED TO – DR. B.M GURUPADYYAY M.PHARM, Ph.D DEPARTMENT OF PHARMACEUTICAL CHEMISTRY   1 SUBMITTED BY- SHALIN SHIKHAR M.PHARM(1 ST YEAR) DEPARTMENT OF PHARMACEUTICS

2. CONTENTS PRINCIPLE OF ELECTROPHORESIS PRINCIPLE OF PAPER ELECTROPHORESIS APPLICATION OF PAPER ELECTROPHORESIS SEPERATION OF AMINO ACIDS SEPERATION OF PROTEIN SERUMS STUDY OF SICKLE CELL ANAEMIA IN HEMOGLOBIN SEPERATION OF ANTIBIOTICS CAN BE CARRIED OUT OTHER USES REFERENCES 2

3. ELECTROPHORESIS PRINCIPLE- Under the influence of an electric field charged molecules and particles migrate in the direction of the electrode bearing the opposite charge . Because of their varying charges and mass, the different molecules and particles of a mixture will migrate at different velocities and thus will thus be separated into single fractions. 3

4. PAPER ELECTROPHORESIS PRINCIPLE A mixture of ions or ionisable substance is applied on the centre of a paper, previously immersed in a buffer of known ionic strength. This paper is placed across two trays, filled with buffer , into which two electrodes are immersed. When a voltage is applied across these electrodes, the ions or ionisable substance migrate towards anode and cathode, based on their charges. 4

5. Applications- 5

6. SEPERATION OF AMINO ACIDS 6

7. SEPARATION OF AMINO ACIDS AT NEUTRAL SOLUTION: The acidic amino acids will have a net positive charge and will migrate toward the cathode(- ve ) The basic amino acids will have net – ve charge and migrate toward the anode(+ pole).  Electrically neutral amino acids or the zwitter ion will not migrate and they will stay at the origin . N inhydrin is added on the spot of amino acids. 7

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9. EFFECT OF INCREASE AND DECREASE OF THE PH IN THE AMINO ACID Amino acid in basic solution If we increase the pH of a solution of an amino acid by adding hydroxide ions, the hydrogen ion is removed from the -NH 3 +  group. The amino acid would be found to travel towards the anode the positive electrode. 9

10. MECHANISM Amino acid in acidic solution If we decrease the pH by adding an acid to a solution of an amino acid, the -COO -  part of the zwitter ion picks up a hydrogen ion. This time, during electrophoresis, the amino acid would move towards the cathode (the negative electrode). Amino acids as zwitterions An amino acid has both a basic amine group and an acidic carboxylic acid group. 10

11. SEPERATION OF PROTEINS IN SERUM The serum proteins move through the fine cellulose mesh of the paper under the influence of an electric current The separation of the various fractions being brought about by differences in their electrophoretic mobility at a selected P h After separation, the protein bands are fixed in position  and stained with a protein-specific stain, such as Amido Black, and destained . Usually five separate fractions are seen: one albumin and four globulin fractions, designated ALPHA 1,ALPHA2, GAMMA and BETA. 11

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13. STUDY OF SICKLE CELL ANAEMIA All varieties of hemoglobin moved to the anode with varying degrees of mobility. An adult hemoglobin migrates with greater mobility than any other hemoglobin as it has the highest number of positive charge. Sickle cell(trait) hemoglobin has fewer positive charge as compared to the normal adult hemoglobin, but more positive charge as compared to sickle thalassemia, so it is intermediate. NORMAL HEMOGLOBIN> SICKLE CELL(TRAIT)> SICKLE THALASSEMIA>SICKLE CELL 13

14. a- Normal Adult Hemoglobin(A*:100 & S*:0) b- Sickling Trait( A :65%& S: 35%) c- Sickle thalassemia ( A:18% & S: 82%) d- Sickle Cell Anemia(A:0 % S: 100%) A- NORMAL HEMOGLOBIN , S- SICKLING HEMOGLOBIN 14

15. SEPARATION OF ANTIBIOTICS IN DIFFERENT SAMPLES Paper Electrophoresis is useful in separating water-soluble antibiotics, although it takes more time and mobility of the sample may be influenced by the inorganic salts. It is found that high voltage paper electrophoresis for 15 minutes , give a good separation of antibiotics especially water soluble basic or amphoteric antibiotics even with crude preparations. Antibiotics on the are detected by the process of bioautography * using test organisms such as Straphylococus aureus , Micrococus flavus , Bacillus subilis or Eschericha coli. Ninhydrin and Sakagauchi Reaction* and ultraviolet light is used for the detection of the antibiotics . * Bioautography  is a laboratory technique to detect substances affecting the growth rates of test organisms in complex mixtures and matrices . * Sakagauchi Reaction is a chemical test used for detecting the presence of arginine in proteins. 15

16. OTHER USES Paper electrophoresis is also used in the separation and identification of alkaloids. P.E can also be used for testing the suitability of Municipal water supplies, toxicity of water and other environmental components. Drug-testing industry uses paper electrophoresis to determine presence of illegal or recreational drugs in job applicants and crime suspects.  PE has been used since 1950s by the investigators and in forensics to analyze inks used in currency to check the counterfeiters. 16

17. REFERENCE - Text Book of Pharmaceutical Analysis, DR.S.RAVI SANKAR Electrophoresis In Practice, REINER WESTERMEIER Analytical Separation Of Amino Acids By Paper Electrophoresis(Research Article), GLADYS N. ATFIELD and C.J.O. R. MORRIS Paper Electrophoresis Of Abnormal Hemoglobins and Its Clinical Applications(Research Article, ARNO G. MOTULSKY, MILTON H PAUL, And E.L.DURRUM Lawrence Chasin and Deborah Mowshowitz    Department of Biological Sciences   Columbia University   New York, NY 17