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Electrophoresis and Electrochromatography

Electrophoresis and Electrochromatography. Outlines . An overview of electrophoresis Instrumentation, performance of CE Capillary electrochromatography Electrophoretic techniques, application of electrophoresis. Electrophoresis . Principle:

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Electrophoresis and Electrochromatography

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  1. Electrophoresis and Electrochromatography TJH&NNH

  2. Outlines • An overview of electrophoresis • Instrumentation, performance of CE • Capillary electrochromatography • Electrophoretic techniques, application of electrophoresis TJH&NNH

  3. Electrophoresis • Principle: • separation mainly of charged materials by differential migration across a surface or through a column in an applied potential gradient/electric field; migration rates dependent upon size, shape and charge of species • In the absence of other effects, cations migrate toward the electric field’s negatively charged cathode, and anions migrate toward the positively charged anode. • Ions with higher charge-to-size ratio migrate at a faster rate • Neutral species do not experience the electric field and remain stationary TJH&NNH

  4. Instrumentation • Traditional electrophoresis: • paper, cellulose acetate or polymeric gels used as a supporting medium for the electrolyte solution; enclosed tank with electrodes and buffer reservoirs; dc power supply • Capillary electrophoresis: • narrow-bore fused-silica capillary tube; injection system; detector; recorder or VDU TJH&NNH

  5. Classical gel electrophoresis • Slab gel TJH&NNH

  6. Classical gel electrophoresis • Tube gel TJH&NNH

  7. Capillary electrophoresis (CE) • Power supply (for applying electric field) • Anode & cathode compartments containing reservoirs of the buffer solution • Sample vial containing sample • Capillary tube • detector TJH&NNH

  8. Flow through capillary is result of superposition two processes: • Flow through capillary is result of superposition two processes: TJH&NNH drag

  9. Electroosmotic flow • Definition: The overall solvent movement caused by positively-charged ions migrating towards the negative electrode and carrying solvent molecules in the same direction. • Cause: electrical double layer that develops at the silica/solution interface TJH&NNH

  10. Process • At pH > 3, the inside wall of a silica capillary is negatively charged owing to ionization of the surface silanol groups (Si-OH) TJH&NNH

  11. Buffer cations congregate in an electrical double layer adjacent to the negative surface of the silica capillary TJH&NNH

  12. The cations in the diffuse outer layer to the double layer are attracted toward the cathode, or negative electrode, and since they are solvated, they drag the bulk solvent along with them TJH&NNH

  13. Detectors for CE TJH&NNH

  14. Applications of CE • Separation of small ions • Separation of molecular species (small synthetic pesticides, pharmaceuticals that are ions or can be derivatized to yield ions) TJH&NNH

  15. Capillary electrochromatography • CEC is a relatively new techniques • A hybridof capillary electrophoresis and high-performance liquid chromatography, combining elements of both • A fused silica CE column is packed with an HPLC stationary phase, usually a bonded-phase silica (1.5-3µm), and filled with a buffer solution (>pH4). TJH&NNH

  16. Under the influence of an applied voltage, an electro-osmotic flow (EOF) of buffer towards the cathodic end of the capillary is generated. • The flow of mobile phase has a flat profile, as in CE, but unlike in HPLC there is no pressure drop because the driving force is generated throughout the length of the column. • Separations are based on both electrophoretic migration for charged analytes and chromatographic sorption for neutral species, hence the name capillary electrochromatography. TJH&NNH

  17. CEC TJH&NNH

  18. Electrophoretic techniques • Capillary zone electrophoresis (CZE) • Electrolyte migrates through the capillary • Most widely used • Samples are applied as a narrow band that is surrounded by the electrolyte buffer • Application: ionic spesies • Micellarelectrokinetic capillary chromatography (MEKC) • A cationic or anionic surfactant is added in excess to the background electrolyte to form charged micelles • Micelles (immiscible in the solution) form a pseudo-stationary phase analogous to the stationary phase in HPLC • Application: chiral analyses, ionic substances & neutral compounds TJH&NNH

  19. Electrophoretic techniques • Capillary gel electrophoresis (CGE) • Transposition of the classic agarose or polyacrylamide gel electrophoresis into a capillary • Capillary filled with an electrolyte impregnated into a gel • Application: DNA sequencing • Capillary isoelectric focusing (CIEF) • Creating a stable & linear pH gradient in a surface-treated capillary which has been filled with a solution that contains ampholytes (zwitterions) • Application: characterization of protein mixtures, determination of protein isoelectric points TJH&NNH

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