Electrophoresis (1) PDF

ELECTROPHORESIS AND ELECTROCHROMATOGRAP HY  Electrophoresis—An Overview Definition: The differential movement for migration of ions by attraction or repulsion in an electric field. – Separation of components of a mixture using an electric field v=Eq/f...

ELECTROPHORESIS AND ELECTROCHROMATOGRAP HY  Electrophoresis—An Overview Definition: The differential movement for migration of ions by attraction or repulsion in an electric field. – Separation of components of a mixture using an electric field v=Eq/f – v = velocity of molecule – E = electric field – q = net charge of molecule – f = friction coefficient Principle of electrophoresis  Electrophoresis is the process of moving charged molecule in solution by applying an electric field across the mixture + - Electrode - + + - + + - - - - + + + - 6 Principle of Separation According to charge According to size 7 (1) According to charge  When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge. 8  (2) According to size 1 2 Mice run faster through the forest than elephant9 10  Types of Electrophoresis Capillary Native Polyacrylimide Gel Electrophoresis (PAGE) SDS-PAGE Slab Paper  Instruments and Reagents Buffer Power supply Supporting media Detection and Quantification 12  I. Buffer Function of buffer – 1. carries the applied current – 2. established the pH – 3. determine the electric charge on the solute High ionic strength of buffer – produce sharper band – produce more heat Commonly used buffer Barbital buffer & Tris-EDTA for protein Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8) 13  II. Power Supply Function: To supply electric power – Constant voltage (increasing of heat over time) Heat = E x I x t E= EMF in volts (V) – Constant current I = Current in amperes (A) according to Ohm’s law E= I x R t = Time in second (s) R decrease over time, which inturn decrease the heat effect 14 III.Support materials used in electrophoresis Paper Starch Agar/agarose Cellulose acetate polyacrylamide gel 15 Electrophoretic methods can be subdivided into two :categories, depending upon whether the separation is carried out in the absence or presence of a supporting or stabilizing medium. In the free-solution method, the sample solution is introduced as a band at the bottom of a V-tube filled with a buffered liquid. A field is applied by means of electrodes located near the tube ends; the differential movement of the charged particles toward one or the other electrode is observed. Separations occur as a result of differences in migration rates; these rates in turn are related to the charge-to-mass ratios and the inherent mobilities of the species in the medium. The 16  Experimental Methods of Electrochromatography The solid media employed in electrochromatography are as numerous and varied as those encountered in the other chromatographic methods. Examples include paper, cellulose acetate membranes, cellulose powders, starch gels, ion- exchange resins, glass powders, and agar gels. Depending upon the physical nature of the solid, separations are performed on strips of paper or membranes, in columns, in trays, or in thin layers supported by glass or plastic. Despite certain disadvantages, filter paper and cellulose acetate are the most widely used Figure is a schematic diagram illustrating three of the many ways for performing a paper electrophoresis. In Figure , a strip of paper is held horizontally between two containers filled with a buffer mixture; the paper is well soaked with buffer and evaporation is prevented by housing the apparatus in an air-tight container. The sample is introduced as a band at the center of the strip, and a dc potential of 100 to 1000 V is applied across the two electrodes. The latter are sufficiently isolated from the paper to prevent the electrode reactions from altering the composition of the buffer on the paper. Currents in the milliampere range are ordinarily observed. After a suitable electrolysis period, the The inverted V arrangement shown in Figure b is also widely used. Here, the sample is introduced at the apex of the V; cationic species migrate down one arm of the V and anionic constituents down the other. Many other modifications of the apparatus shown in Figure b have been described, and several are available commercially. In some, the paper is supported (either horizontally or at some suitable angle) between two glass or plastic plates; the plates may be cooled to dissipate the heat generated by the current. In all of these devices, the rate at which components migrate is controlled primarily by their charge-to-mass ratios and their mobilities, with adsorption or other equilibria having only a secondary influence. Figure c illustrates an apparatus for a type of two- dimensional electrochromatography. Here, the paper is held vertically and the sample components are carried down the sheet by the flow of a buffered solvent. Separation then occurs in the