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Moving boundary electrophoresis mpat

Fully described view of moving boundary electrophoresis, useful for pharma pg of all divisions.

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Moving boundary electrophoresis mpat

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  2. 2. ELECTROPHORESIS: The word electrophoresis is derived from Greek word, which means, “Born by electricity”. It is the migration of charged particles or molecule in a medium under the influence of an applied electric field. Under the influence of electric field charged particles migrate towards anode (+) or cathode (-). 2
  3. 3. The usual purposes are:- This technique involves the observation of motion of small particle in an electric field with a microscope. e.g. RBC, neutrophils, bacteria etc. The ocular micrometer can be used to measure the electrophoretic mobility. Used for measuring zeta potential of R.B.C., neutrophils, bacteria. 3
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  5. 5. The moving boundary method was the first used by Tiselius to demonstrate the efficacy of the electrophoretic process. This method allows the charged species to migrate in a free moving solution in the absence of a supporting medium. Samples are fractioned in a U shaped tube that has been filled with unstabilized buffer. An electrical field is applied by means of electrodes at the ends of the U tube. Separation takes place as a result of difference in mobilities. 5
  6. 6. Apparatus consists of U tube, with electrodes located at the two ends used to apply an electric field. The lower part of the cell is filled with lyophillic solution under examination, sometimes the sample solution is introduced into the bottom of the U tube through a 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. For this purpose, the apparatus is placed in constant temperature bath at 4°c. 6
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  8. 8. 8 MECHANISM OF ELECTROPHORESIS
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  10. 10. 10 Anionic species can be separated by using a narrow-bore tube as the separation chamber. It is connected with Anode and Cathode compartment Anode compartment and narrow-bore tube are filled with an electrolyte Sample is introduced into the Cathode compartment Anionic species migrate towards the anode and sample anions can never pass the anionic species of the leading electrolyte because its effective mobility is higher. Mobilities of the anionic species of the sample differ, however, so that some of them will migrate forward.
  11. 11. contd 11 Substance B mixed with C, forms the second sample zone. The third zone contains the mixture A+B+C. Substance A which is more mobile than the other substance of the sample is partially separated from B & C
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  13. 13. In Moving Boundary Electrophoresis, Zones generally contains more ionic species of the sample. Composition of the sample plays an important role in the determination of the concentrations, pH values and conductivities of the different zones. So Effective mobilities can be measured by using Moving boundary electrophoresis. 13
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  15. 15. The rate of migration (Separation of particles) during electrophoresis will depend on the following factors: 1. The Sample 2. The Electric Field 3. The Buffer 4. Electrolysis 15
  16. 16. Charge/mass ratio of the sample determines its electrophoretic mobility. The mass consists of not only the size (molecular weight) but also the shape of the molecule. a)Charge: The higher the charge, greater is the electrophoretic mobility. The charge is dependent on pH. b) Size: The bigger molecules have a small electrophoretic mobility compared to the smaller particles. c) Shape: The globular protein will migrate faster than the fibrous protein 16
  17. 17. 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. 17
  18. 18. 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… 18
  19. 19. contd The choice of buffer depends upon the type of sample being electrophoresed. b) Ionic Strength: “Ionic 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. 19
  20. 20. 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. 20
  21. 21. 21 Biologically active fractions can be removed without the use of denaturing agents. A reference method for measuring Electrophoretic mobilities. Minute concentration of the sample can be detected upto 0.05mg/ml by Interferometric optical system
  22. 22. 22 The resolution of the technique is very low due to the mixing of the sample as well as over- lapping of the sample components. The electrophoresis technique is not good to separate and analyze the complex biological sample instead it can be used to study the behavior of the molecule in an electric field.
  23. 23. To study the homogenecity of a macromolecular system. It is employed in biochemical and clinical fields i.e in the study of protein mixtures such as blood serum, haemoglobins and in the study of antigen-antibody interactions. It is also used for the separation of scopolamine and ephedrine using buffer with pH 4.2 Protein research/purification. 23
  24. 24. 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 l Dekker, Newyork, pg no: 850-865 24
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