Electrophoresis

1. Welcome To my presentation

2. 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

3. electrophoresis

4. 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) Figure 1: Motion of a charged particle by electrophoresis * * A useful method to separate substances based on their charge – to – mass ratios

5. 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 = μ e E w here, v = velocity of an ion E = electric field strength (Vcm -1 ) μ e = electrophoretic mobility = distance migrated in a certain time period The electrophoretic mobility is given by μ e = (when electric force = frictional drag) showing that small highly charged species have high mobility and vice versa.   * *

6. Driving force of migration : * R esultant 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. F igure 2: Illustration of electrophoresis retardation

7. 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 l ow solvent capacity e nhancing the resolution Gels 3 dimensional semisolid colloids r esolving power enhanced due to sieve effect operating p repared from starch, agar, or polyacrylamide *

8. General procedure for electrophoresis : Figure 3: Fundamental of electrophoresis

9. 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

10. Types of electrophoresis : Figure 4: Types of electrophoresis

11. Techniques of electrophoresis: Figure 5: Different techniques of electrophoresis

12. Low voltage electrophoresis : * Two compartments to hold the buffer and electrodes Figure 6: Apparatus for low voltage electrophoresis * A suitable carrier for support medium e nding 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

13. 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: E lectrophoretogram of plasma proteins on cellulose acetate at pH 8.6

14. 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

15. Capillary electrophoresis (CE) : * Separation of analyte species achieved on the basis of differential migration in an electric field through narrow bore fused silica capillary columns (25 – 100 μ m). Figure 9: Separation modes of capillary electrophoresis

16. 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

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

18. 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

19. 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.

20. Thank you