Capillary Electrophoresis

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Capillary Electrophoresis

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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

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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.

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Types of Electrophoresis

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

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Capillary Electrophoresis Apparatus
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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

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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

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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

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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

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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

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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

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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

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Ooooo! It MOVES
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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

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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

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Equipment
Yowzah!!!

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

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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

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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

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ITS TIME TO PARTY!!!