electrophoresis ppt


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ELECTROPHORESIS Prepared by : Maulik Oza M.Pharm (Quality Assurance) Roll no : 6 Guided by : Mr.Dharmendrasinh A. Baria Assistant Professor M.Pharm (Pharmaceutical chemistry) Dharmaj Degree Pharmacy College Dharmaj 1


ELECTROPHORESIS When a potential difference is applied between the electrodes in a colloidal solution ,colloidal particles are carried to either positive or negative electrode. In short they are electrically charged with reference to dispersion medium. Migration of charged solutes in a liquid medium under an electrical field Many biological molecules have ionisable groups eg. amino acids, proteins, nucleotides, nucleic acids 2

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Under an electric field charged particles migrate to anode (+) or cathode (-) Migration of individual ions as well as to colloidal aggregate . Rate of travel of the partical depend on Characteristics of the particals. Properties of the electrical field. Temperature. Nature of the suspended medium 3

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Application for the purification Preparation & measurement. Electrophoresis methods are powerful analytical tool for fractionating component of mixture are aggregate or monodispersed. 4


TYPES OF ELECTROPHORESIS FREE SOLUTION ELECTROPHORESIS Moving boundary electrophoresis Density gradient electrophoresis ZONE ELECTROPHORESIS Paper electrophoresis Gel electrophoresis Capillary zone electrophoresis Immuno electrophoresis 5

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FREE SOLUTION ELECTROPHORESIS Absence of supporting & stabilizing medium ZONE ELECTROPHORESIS Presence of supporting & stabilizing medium eg. Paper 6

Principle: :

Principle: Movement of charged particles when suspended or dissolved in a polar liquid and the mixture is placed in an electric field. Movement of charged particles in gases, although with modifications the basic concepts could be applied to these suspensions. For most purposes the movement of the ion is translational and carried out under the influence of a constant electric field. 7


THEORY When a constant electric field is applied across a column of water, current flows and gases evolve at the electrodes hydrogen at the cathode and oxygen at the anode. This phenomenon is known as electrolysis. The experimental basis for these laws of conduction of solutions involved the measurement of conductivity of a solution electrochemical equivalences showing that the conductance of a solution is the product of the number of ion in the solution, the charge carried by each ion, and the velocity or their mobilities u ±. 8

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Quantitative relationships were developed between the current i carried through the solution by C + gram moles of a univalent anion and the mobilities observed in an electricfield E : i + = u + C + E . (1) The equivalent expression for the cat ion is the same as Eq. (1), and so the total current i is i = i + + i − = ( u + C + + u − C −) E , (2) which can be compared directly with define resistance, E = 1 /k, (3) Where k is the conductance, hence, k = u + C + + u − C − and the conductance is proportional to the velocities of the ions. 9

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FIGURE Idealized electrophoresis experiment in which boundaries between three salts are formed initially at a and b . After passage of a known quantity of electricity, the boundaries have moved to a and b . 10

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It by forming a suitable boundary between the ion and the solvent and measuring the velocity of this boundary given in Fig. . A solution of a simple salt, KCl , separates An elementary arrangement for making this experiment is two solutions of other salts, LiCl in the anode compartment and potassium acetate in the cathode compartment. 11

Moving boundary electrophoresis :

Moving boundary electrophoresis 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 unstabilised buffer An electrical field is applied by means of electrodes at the ends of the tube Separation takes place as a result of difference in mobilities Bands are obtained which are located by optical systems 12

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Sample can be large ,so long as the boundary,or long edge, between sample & buffer is kept sharply defined. When such a prepation is subjected to electrophoresis, the boundary of each component retains its identity. Component move at rates proportional to their mobilities . As a result, a pattern is developed in which components overlap to make sharply defined steplike contribution to the total solute concentration. Each component within the tube is visualised as a separate cylinder of solute migrating in an independent manner. 14

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Cylinders move at different rates, so sharp changes in concentration occur at the points where successive fraction overlape . The boundaries of the fraction can be detected by scanning the tube with a refractometer,because refractive index of a solution is directiy proposional to the concentration of the solute. The conc. of each fraction will be calculated as a function of its contribution to the change in refractive index. This is called as moving boundry electrophoresis. The fundamental difference between the two methods is that zone electrophoresis uses an anticonvection medium boundry electrophoresis does not. 15

Reference :

Reference Encyclopedia of Physical science and Technology Third Edition Analytical Chemistry S. P. Spragg Pages 363-378. Instrumental analysis by b.k . sharma 3 th edition page no c-269-273. 16

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Thank You 17

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