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Shahjalal University of Science & Technology, Sylhet Department of Chemistry Course No. : CHE 300 Course title : Seminar and Oral A presentation on Electrophoresis Presented by, Tanjila Islam Reg. No. : 2010131019 Semester : 3/2

electrophoresis

Basic of electrophoresis : Differential rate of migration of ion molecule in an electrolyte solution under the influence of an applied electric field in a support medium (e.g. paper, gel or capillary tube) * A useful method to separate substances based on their charge – to – mass ratios * Figure 1: Motion of a charged particle by electrophoresis

Principle : * Charged ion or molecule migrates when placed in an electric field Rate of migration depends on its net charge, size, shape and the applied electric current v = μeE where, v = velocity of an ion E = electric field strength (Vcm-1) μe = electrophoretic mobility = distance migrated in a certain time period The electrophoretic mobility is given by μe = q 6πηr (when electric force = frictional drag) showing that small highly charged species have high mobility and vice versa. * *

Driving force of migration : * Resultant of the electrostatic force of attraction between the electric field and the charged molecule, and the retarding forces due to friction and electrostatic repulsion from molecules of the transport medium. Figure 2: Illustration of electrophoresis retardation

Supporting media for electrophoresis : * Paper filter paper such as Whatman no.1 and no.3MM Used to good effect * Cellulose acetate - containing 2 to 3 acetyl groups - to give sharper bands - more easily rendered transparent low solvent capacity enhancing the resolution * Gels 3 dimensional semisolid colloids resolving power enhanced due to sieve effect operating prepared from starch, agar, or polyacrylamide

Separation of different ions migrating at different speeds General procedure for electrophoresis : Immersion of two electrodes in two separate buffer chambers but not fully isolated from each other Migration of charged particles from one chamber to the other by using an electric field Separation of different ions migrating at different speeds Figure 3: Fundamental of electrophoresis

Factors affecting electrophoretic mobility : * Charge – higher the charge greater the mobility * Size – bigger the molecule greater the frictional and electrostatic forces exerted on it by the medium i.e. larger particles have smaller electrophoretic mobility compared to smaller particles Electric field – increase of migration with the increase of voltage gradient * Buffer – dependence of migration on pH of the buffer * * Ionic strength – greater the ionic strength of the buffer solution higher proportion of the current hence electrophoretic mobility

Types of electrophoresis : Frontal Micro electrophoresis Moving boundary Zone electrophoresis Paper electrophoresis Cellulose acetate electrophoresis Gel electrophoresis Figure 4: Types of electrophoresis

Techniques of electrophoresis: Low voltage (LVE) High voltage (HVE) SDS polyacrylamaide gel (SDS-PAGE) Isoelectric focusing Immunoelectrophoresis Discontinuous electrophoresis Figure 5: Different techniques of electrophoresis

Low voltage electrophoresis : Two compartments to hold the buffer and electrodes * A suitable carrier for support medium ending in contact with the buffer medium * To provide voltage gradient ̴ 5 Vcm-1, a power pack supplying up to 500 V or even 1000 V and 0 – 150 mA * Figure 6: Apparatus for low voltage electrophoresis

Application of LVE : To separate any ionic substances * The examination of biological and clinical specimens for amino acids and proteins * * Separation of sugars Figure 7: Electrophoretogram of plasma proteins on cellulose acetate at pH 8.6

High voltage electrophoresis : * To obtain voltage gradients up to 100 Vcm-1, high voltage and current supplying * Using cooling plates for heat dissipation generated by high voltage * Less than of 1h analysis time * Working best with small ions deriving from small peptides and amino acids Figure 8: HVE apparatus

Capillary electrophoresis (CE) : zone electrophoresis (CZE) Capillary gel electrophoresis (CGE) Isoelectric focusing Isotachophoresis (ITP) Miscellar electrokinetic capillary chromatography (MECC) Separation of analyte species achieved on the basis of differential migration in an electric field through narrow bore fused silica capillary columns (25 – 100 μm). * Figure 9: Separation modes of capillary electrophoresis

Overview of instrumentation of CE : * A fused capillary column dipping into two electrolyte buffers containing Pt foil cathode or anode across 15 – 60 kV voltage applied Introducing a small volume of sample at one end of capillary * * Migration of sample through the capillary under the force of applied electric field Figure 10: Schematic of a capillary zone electrophoresis

Advantages of CE : * Power dissipation minimized by high electrical resistance Having voltage gradients up to 100 – 500 Vcm-1 necessary for rapid separations * * Most prominently used because of its faster results and high resolution separation * Large range of detection methods available * No Joule–Thompson effect * No band broadening

Applications of electrophoresis : * DNA analysis Protein analysis * Antibiotic analysis * * Vaccine analysis Detection of damaged genes by gel electrophoresis * * To use in forensic research Figure 11: A simple view of protein separation

Conclusion : Although not in principle a chromatographic method, electrophoresis used in conjunction with paper chromatography and gel materials, proves an extremely useful method for separation of charged substances, ranging from small ions to large charged macromolecules, of biological and biochemical interest. * It is widely used yet it has some limitations. *

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