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Capillary Electrophoresis

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Title: Capillary Electrophoresis


1
Capillary Electrophoresis

2
ElectrophoresisAn 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
  • vEq/f
  • v velocity of molecule
  • E electric field
  • q net charge of molecule
  • f friction coefficient

3
Electrophoresis- overview cont.
  • Can determine the size, shape, and charge of a
    molecule
  • Different forms of electrophoresis are used for
    each of these factors independently or in
    combination.

4
Types of Electrophoresis
  • Capillary
  • Native Polyacrylimide Gel Electrophoresis (PAGE)
  • SDS-PAGE
  • Slab
  • Paper

5
Capillary Electrophoresis Apparatus
6
Capillary Electrophoresis The Basics
  • Electrophoresis in a buffer filled, narrow-bore
    capillaries
  • Each capillary is about 25-100 µm in internal
    diameter
  • When a voltage is applied to the solution, the
    molecules move through the solution towards the
    electrode of opposite charge
  • Depending on the charge, the molecules move
    through at different speeds
  • Separation is achieved

7
Basics cont.
  • A photocathode is then used to measure the
    absorbencies of the molecules as they pass
    through the solution
  • The absorbencies are analyzed by a computer and
    they are represented graphically

8
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9
Electrophoretic Mobility
  • The movement of ions solely due to the electric
    field, potential difference
  • Cations migrate toward cathode
  • Anions migrate toward anode
  • Neutral molecules do not favor either

10
Electrophoretic Mobility
  • vEq/f
  • vep µepE
  • µ q/(6p?r)
  • ? is buffer viscosity
  • r is solute radius
  • Properties that effect mobility
  • Voltage applied
  • Size and charge of the solute
  • Viscosity of the buffer

11
Electroosmotic Flow
  • As the buffer sweeps toward the anode due to the
    electric field, osmotic flow dictates the
    direction and magnitude of solute ion flow within
    the buffer
  • All ions are then swept toward the anode.
  • Negative ions will lead the neutral ions toward
    the anode
  • Positive ions will trail the neutral ions as the
    cathode pulls them

12
Electroosmotic Mobility
  • veof µeofE
  • µeof ?? / (4p?)
  • ? buffer dielectric constant
  • ? zeta potential
  • Zeta Potential
  • The change in potential across a double layer
  • Proportional to the charge on the capillary walls
    and to the thickness of the double layer.
  • Both pH and ion strength affect the mobility

13
Total 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

14
Ooooo! It MOVES
15
Equipment
  • Capillary tube
  • Varied length but normally 25-50 cm
  • Small bore and thickness of the silica play a
    role
  • Using a smaller internal diameter and thicker
    walls help prevent Joule Heating, heating due to
    voltage

16
Equipment
  • Because ions are in the bulk solution are about
    to travel through the capillary without
    interference from the capillary itself, there is
    no dramatic drop in potential within the
    capillary
  • No meniscus is made

17
Equipment
Yowzah!!!
  • Detector
  • UV/Visible absorption
  • Fluorescence
  • Radiometric (for radioactive substances)
  • Mass Spec.

18
Applications
  • Analysis of carbohydrates
  • Analysis of inorganic anions/metal ions
  • DNA profiling
  • Protein identification
  • Advantages
  • Fast
  • Small Sample
  • Relatively inexpensive
  • Automated
  • Disadvantages
  • Cannot identify neutral species
  • Joule Heating
  • Cannot discern shape

19
Resources
  • Altria, Kevin. http//www.ceandcec.com
  • Hardy, James K. University of Akron.
    http//ull.chemistry.uakron.edu/chemsep/electropho
    resis/
  • 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_C
    LAS_2002_Fall_CHEM451-2/displaymaterialsLect5-SDS
    -PAGE.ppt/SESSION10480818522765085466263962826/L
    ect5-SDS-PAGE.ppt

20
ITS TIME TO PARTY!!!
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