moving boundary electrophoresis6

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CONCEPT OF MOVING BOUNDARY ELECTROPHORESIS : 

PRESENTED BY:- BHATT BHAVIK R. 1ST M. PHARM. DEPT. OF PHARMACEUTICS NGSMIPS CONCEPT OF MOVING BOUNDARY ELECTROPHORESIS

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INTRODUCTION FREE ELECTROPHORESIS MOVING BOUNDARY ELECTROPHORESIS REFERENCES

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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 an electric field charged particles migrate to anode (+) or cathode (-).

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The usual purposes are:-

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This technique involves the observation of motion of small particle in an electric field with a microscope. e.g. RBC, neutrophils, bacteria etc. The suspension is contained in a closed system composed of a thin walled section for optical observation and of suitable electrode compartment. The ocular micrometer can be used to measure the electrophoretic mobility. Use for measuring zeta potential of R.B.C., neutrophis, bacteria.

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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 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.

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PRINCIPLE This method allows the charged species to migrate in a free moving solution without supporting medium. In a free solution, there is minimal friction resistance between ions and solution. Separation was observed by bands of wavy patterns in the solution.

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Initial formation of sharp boundaries. Effective thermostatic control to minimize conventional boundary disturbances due to the heating effect of the applied current. Large electrode vessels containing reversible electrodes. An optical system for following the movements of boundaries.

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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.

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Fig. apparatus used in electrophoresis Fig. mechanism of electrophoresis

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Buffered solution of macromolecules is placed under a layer pure buffer solution. The whole cell may then be immersed in a constant temperature bath and electric field between the electrode is generated. Macromolecules bear a net –ve charge move towards the anode & +ve charge move towards the cathode. This movement not give a better resolution. pH of buffer solution is therefore chosen that all the macromolecules bear a net –ve charge. When the electric field is generated between the electrode, the movement will be toward the anode.

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So they migrate from the macromolecule solution to the pure buffer solution and from a boundary. As a result, there is a small change in reflective index of the solution. At this boundary the mode of refraction of macromolecule is different from that of pure buffer. The RI changes can be measured by appropriate optical device. The concentration gradient which are formed during electrophoresis is usually detected by Schlieren optical method.

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ELECTROPHORETIC MOBILITY REFRACTIVE INDEX

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The technique is used for measuring zeta potential of cells such as RBC, neutrophils & bacteria. Biological and biochemical research, pharmacology, clinical investigation. DNA analysis. In protein and enzyme analysis. For examining homogenecity of macromolecule system. To study colloidal dispersion.

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Dely z., Electrophoresis , Elsevier scientific publishing company, p.156-158 Scoot R. P. W., A Text book of Isotachophoresis, page no: 345-352 Sharma BK. Instrumental methods of chemical analysis. Goel publishing house, Meerut; 20th edition 2001. pg no: 113-21. Robinson J. W., Undergraduate instrumental analysis, 6th edition, 2009, Marcel Dekker, Newyork, pg no: 850-865

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