M.Pharm. Pharmaceutical Analysis Moving Boundary Electrophoresis.pptx

M.Pharmacy- Pharmaceutical Analysis-Moving Boundary Electrophoresis Presented by Dhanashree Kolhekar
The word electrophoresis is derived from Greek word ,which means, “born by electricity”
Electrophoresis is defined as the migration of charged particle through a solution under the influence of an external electric field.
Ions that are suspended between two electrodes tends to travel towards the electrodes that posses opposite charges.

1. Moving Boundary Electrophoresis Presented By DHANASHREE GIRIDHAR KOLHEKAR 1 st SEM/ 1 st YEAR M.Pharm (Pharmaceutical Analysis) Babasaheb Bhimrao Ambedkar University Lucknow, Uttar Pradesh.

2. CONTENTS INTRODUCTION PRINCIPLE TYPES OF ELECTROPHORESIS MOVING BOUNDARY ELECTROPHORESIS INSTRUMENTATION FACTOR AFFECTING SEPARATION APPLICATION Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

3. INTRODUCTION The word electrophoresis is derived from Greek word ,which means, “born by electricity” Electrophoresis is defined as the migration of charged particle through a solution under the influence of an external electric field. Ions that are suspended between two electrodes tends to travel towards the electrodes that posses opposite charges. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

4. PRINCIPLE OF ELECTROPHORESIS The fundamental principle of electrophoresis is that any charged ion or molecule migrates towards oppositely charged electrode under the influence of external electric field . The rate of migration of these ions depends on its charge , size and the applied electric field. Electrophoresis is one of techniques in the laboratory , which is used to separate RNA,DNA molecules and protein based on their electric charge and size. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

5. TYPES OF ELECTROPHORESIS Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

6. MOVING BOUNDARY ELECTROPHORESIS Moving boundary electrophoresis is electrophoresis is electrophoresis in free solution. It was developed by Arne Tiselius. The principle of moving boundary electrophoresis is that there is motion of charged particle through stationary liquid under the influence of electric field. This method allows the charged species to migrate in a free moving in a free moving solution in absence of supporting medium . An electrical field is applied by means of electrodes at the end of U tube. Separation takes place as a result of difference in mobilities. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

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9. Instrumentation Apparatus consist of “U” tube with electrode located at the two ends used to apply on electric field. The lower part of the cells is filled with lyophilic solution under examination sometimes the sample solution is introduced into the bottom of the “U” tube through capillary arm while upper part contains only the buffer solution. Care must be taken to minimize the disturbing effect of convection caused by an increase in temperature during the passage of current through the solution. The apparatus is placed in constant temperature. Detection is done by measuring refractive index throughout the solution. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

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11. ADVANTAGES AND DISADVANTAGES ADVANTAGES Minute concentration of sample can be detected. A reference method for measuring electrophoretic mobility. Biologically active fragments can be recovered without the use of denaturing agents. DISADVANTAGES Expensive Resolution of the techniques is low due to overlapping of sample components. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

12. FACTOR AFFECTING SEPARATION The rate of migration ( separation of particles ) during electrophoresis will depend on following factor- THE SAMPLE THE ELECTRIC FIELD THE BUFFER ELECTROLYSIS Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

13. THE SAMPLE Charge/mass ratio of sample determines its electrophoretic mobility. The mass consist of not only the size (molecular weight) but also the shape of the molecule. Charge : The higher the charge, greater is the electrophoretic mobility. The charge is dependent on pH. Size: The bigger molecules have a small electrophoretic mobility compared to the smaller particle. shape: The globular protein will migrate faster than the fibrous protein. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

14. THE ELECTRIC FIELD The rate of migration under unit potential gradient is referred to as "Mobility of the ion". An increase in potential gradient increases the rate of migration. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

15. THE BUFFER The buffer can affect the electrophoretic mobility of the sample in various ways. The electrophoretic mobility is greatly affected by the pH of the buffer, particularly when the sample is weak acid or a weak base, because the pH establishes its degree of ionization . In case of amphoteric substances such as proteins, the direction of migration depends upon whether the buffer pH is above or below the isoelectric point of the sample substance. a)Composition: Commonly used buffers are "Formate” "Acetate” "Citrate” "Phosphate" etc... Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

16. The choice of buffer depends upon the type of sample being electrophoresed. b) Ionic Strength:" lonic Strength (I) is a measure of the electrical environment of ions in a solution". When increase ionic strength of the buffer means a larger share of the current being carried by the buffer ions & meager (small quantity) proportion carried by the sample ions. When decrease ionic strength, a larger share of the current being carried by the sample ions leading to a faster separation. Note: The ionic strength used is usually between 0.05 to 0.1M. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

17. ELECTROLYSIS Electrophoresis is accompanied by electrolysis which causes microscopic bubbles to accumulate on the electrodes . When a bubble is formed, the electric contact with buffer is lost. Simultaneously, the resistance of the electrode gradually builds up . It can't be overcome by coalesced, once it got coalesced, the bubbles become large enough to break from the surface and as result, contact with buffer is restored. Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

18. APPLICATIONS Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

19. REFERENCES: Dely z., Electrophoresis, Elsevier scientific publishing company, p. 156-158 Sharma BK. Instrumental methods of chemical analysis. Goel publishing house, Meerut; 20th edition 2001. pg no: 113-121 Robinson J. W., Undergraduate instrumental analysis, 6th edition, 2009, marce 1 Dekker, New york , pg no: 850-865 Dhanashree-Kolhekar, M.Pharma (Pharmaceutical Analysis) BBAU, Lucknow

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