2003 capillaryelectrophoresis


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capillary electrophorosis

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2003 capillaryelectrophoresis

  1. 1. CapillaryCapillaryElectrophoresisElectrophoresis
  2. 2. ElectrophoresisElectrophoresis——An OverviewAn Overview• Definition: The differential movement formigration of ions by attraction orrepulsion in an electric field.– Separation of components of a mixtureusing an electric field• v=Eq/f– v = velocity of molecule– E = electric field– q = net charge of molecule– f = friction coefficient
  3. 3. Electrophoresis- overview cont.Electrophoresis- overview cont.• Can determine the size, shape, andcharge of a molecule• Different forms of electrophoresis areused for each of these factorsindependently or in combination.
  4. 4. Types of ElectrophoresisTypes of Electrophoresis• Capillary• Native Polyacrylimide GelElectrophoresis (PAGE)• SDS-PAGE• Slab• Paper
  5. 5. Capillary Electrophoresis ApparatusCapillary Electrophoresis Apparatus
  6. 6. Capillary ElectrophoresisCapillary Electrophoresis –– TheTheBasicsBasics• Electrophoresis in a buffer filled, narrow-borecapillaries• Each capillary is about 25-100 μm in internaldiameter• When a voltage is applied to the solution, themolecules move through the solution towardsthe electrode of opposite charge• Depending on the charge, the moleculesmove through at different speeds– Separation is achieved
  7. 7. Basics cont.Basics cont.• A photocathode is thenused to measure theabsorbencies of themolecules as they passthrough the solution• The absorbencies areanalyzed by a computerand they arerepresented graphically
  8. 8. Electrophoretic MobilityElectrophoretic Mobility– The movement of ions solely due to theelectric field, potential difference– Cations migrate toward cathode– Anions migrate toward anode– Neutral molecules do not favor either
  9. 9. Electrophoretic MobilityElectrophoretic Mobility• v=Eq/fv=Eq/f• vep = μepE• μ = q/(6πηr)μ = q/(6πηr)– η is buffer viscosity– r is solute radius• Properties that effect mobility1. Voltage applied2. Size and charge of the solute3. Viscosity of the buffer
  10. 10. Electroosmotic FlowElectroosmotic Flow• As the buffer sweeps toward the anodedue to the electric field, osmotic flowdictates the direction and magnitude ofsolute ion flow within the buffer– All ions are then swept toward the anode.– Negative ions will lead the neutral ionstoward the anode– Positive ions will trail the neutral ions asthe cathode pulls them
  11. 11. Electroosmotic MobilityElectroosmotic Mobility• veof = μeofE• μeof = ɛζ / (4πη)– ɛ = buffer dielectricconstant– ζ = zeta potential• Zeta Potential• The change in potentialacross a double layer• Proportional to thecharge on the capillarywalls and to thethickness of the doublelayer.– Both pH and ionstrength affect themobility
  12. 12. Total MobilityTotal Mobility• vtot = vep + veof• Migration times– vtot = l/t• l = distance between injection and detection• t = migration time to travel distance l• t = lL/((μep + μeof)V• L = length of capillary• V = voltage
  13. 13. Ooooo!ItMOVES
  14. 14. EquipmentEquipment• Capillary tube• Varied length but normally25-50 cm• Small bore and thicknessof the silica play a role– Using a smaller internaldiameter and thicker wallshelp prevent JouleHeating, heating due tovoltage
  15. 15. EquipmentEquipment• Because ions are in the bulk solution are about to travel throughthe capillary without interference from the capillary itself, there isno dramatic drop in potential within the capillary• No meniscus is made
  16. 16. EquipmentEquipment• Detector– UV/Visible absorption– Fluorescence– Radiometric (forradioactive substances)– Mass Spec.Yowzah!!!
  17. 17. ApplicationsApplications• Analysis ofcarbohydrates• Analysis of inorganicanions/metal ions• DNA profiling• Protein identification• Advantages• Fast• Small Sample• Relativelyinexpensive• Automated• Disadvantages• Cannot identifyneutral species• Joule Heating• Cannot discern shape
  18. 18. ResourcesResources• Altria, Kevin. http://www.ceandcec.com• Hardy, James K. University of Akron.http://ull.chemistry.uakron.edu/chemsep/electrophoresis/• University of Virginia Chemistry Web page.http://www.virginia.edu/chem/people/• http://www.rsc.org/pdf/books/capelectrosc.pdf• Landers, James. Electrophoresis SDS-PAGE.http://toolkit.itc.virginia.edu/cgi-local/tk/UVa_CLAS_2002_Fall_CHEM451-2/displaymaterials:Lect5-SDS-PAGE.ppt/SESSION:104808185227650:85466263962826/Lect5-SDS-PAGE.ppt
  19. 19. ITS TIMETOPARTY!!!