Electrochromatography - A Hybrid Separation Technique

1
Electrochromatography - A Hybrid Separation
Technique

• Gel Filtration Chromatography Capillary
  Electrophoresis Electrochromatography
• info shamelessly taken from Wikipedia
• and
• http//www.unimicrotech.com/products_CEC_instrumen
  t.htm

2
The Idea

• Combine the attributes of size exclusion
  chromatography (gel filtration chromatography)
  with the benefits of gel electrophoresis.
• The two separation mechanisms both operate along
  the length of a gel filtration chromatography
  column which has an electric field gradient
  applied to the column.
• Useful for the separation of large biomolecules
• separated by size due to the gel filtration
  mechanism
• separated by electrophoretic mobility (gel
  electrophoresis)
• Also other chromatographic solute retention
  mechanisms

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The Basics - Gel Filtration or Permeation

• Size exclusion chromatography (SEC)
• particles are separated based on hydrodynamic
  volume
• aqueous mobile phase gel filtration
  chromatography
• organic mobile phase gel permeation
  chromatography
• widely applied for purification and analysis of
  synthetic or bio-polymers (proteins,
  polysaccharides, nucleic acids)
• biopolymers - use a gel stationary phase (usually
  polyacrylamide, dextran, or agarose) at low
  pressures
• synthetic polymers - use either a silica or
  crosslinked polystyrene stationary phase at
  higher pressures
• Various mobile phases can be used

4
The Basics Hydrodynamic Volume

• Related to the radius of gyration - measure of
  the size of an object
• calculated as the r.m.s. distance of the parts
  (or surface) of an object from either its center
  of gravity or an axis
• the radius of gyration is used to describe the
  dimensions of polymer chains
• chain conformations of polymer samples are quasi
  infinite, change over time
• the "radius of gyration" discussed in polymer
• physics must usually be
• understood as a mean over
• all polymer molecules of the
• sample and over time
• Rg determined experimentally with static light
  scattering as well as with small angle neutron-
  and x-ray scattering.
• The hydrodynamic radius is numerically similar,
  and can be measured with size exclusion
  chromatography.

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SEC Illustrated
6
Gel Filtration or Permeation Inst.

• HPLC type setup
• Controller
• Injector
• Liquid mobile phase
• High pressure pumps
• column (size exclusion stationary phase)
• Detector (UV, fluor., or other)
• collector (as waste or fractions)
• Data system (PC)

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Standard Gel Electrophoresis

• Separation uses a gel" as the stationary phase
  it is often a crosslinked polymer
• For proteins or small nucleic acids (DNA, RNA, or
  oligonucleotides) the gel is usually composed of
  acrylamide and a cross-linker (in various ratios)
  producing mesh networks of polyacrylamide with
  different sized pores.
• For larger nucleic acids (greater than a few
  hundred bases), agarose is the preferred matrix.
  
• "Electrophoresis" refers to the electromotive
  force (EMF) that is used to move the molecules
  through the gel matrix.
• the molecules move through the matrix at
  different rates,
• usually determined by mass,
• Motion is toward the positive anode if negatively
  charged or toward the negative cathode if
  positively charged

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The Basics Cap. Electrophoresis

• Capillary electrophoresis (CE), also known as
  capillary zone electrophoresis (CZE)
• used to separate ionic species by their charge
  and frictional forces.
• traditional electrophoresis, electrically charged
  analytes move in a conductive liquid medium under
  the influence of an electric field
• Introduced in the 1960s, the technique of
  capillary electrophoresis (CE) was designed to
  separate species based on their size to charge
  ratio in the interior of a small capillary filled
  with an electrolyte

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The Basics Electrophoretic Mobility

• analyte electrophoretic migration velocity (up)
  toward the electrode of opposite charge is
• up µpE
• µp electrophoretic mobility
• E is the electric field strength
• electrophoretic mobility at a given pH
• z is the net charge of the analyte
• the viscosity (?) of the medium
• r is the Stokes radius of the analyte
• D is the diffusion coefficient.

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The Basics electroosmotic flow

• EOF does not significantly contribute to band
  broadening as in pressure-driven chromatography.
• Capillary electrophoresis separations can have
  several hundred thousand theoretical plates

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The Basics electroosmotic flow

• electroosmotic flow (EOF) of buffer is directed
  toward the cathode (-)
• the electroosmotic flow of buffer gt
  electrophoretic flow of the analytes
• all analytes are carried along with the buffer
  toward the cathode
• analytes do migrate toward the electrode of
  opposite charge
• negatively charged analytes attracted to anode
  (), counter to the EOF
• positively charged analytes attracted to cathode
  (-) with the EOF
• anionic analytes retained longer due to
  conflicting electrophoretic mobilities
• small multiply charged cations migrate quickly
  and small multiply charged anions are retained
  strongly

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The Instrumental Requirements

• Capillary Electrophoresis

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Electrochromatography

• high efficiency of CE is combined with the high
  selectivity of micro-HPLC
• hybrid technique known as capillary
  electrochromatography (CEC).
• utilizes columns similar to those used in
  micro-HPLC
• the mobile phase is driven by an electric
  potential as in CE
• separation mechanism is the result of the
  combination of chromatographic partitioning and
  electrophoretic migration.
• CEC can be done in a CE instrument with a
  micro-HPLC column

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Electrochromatography
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Electrochromatography

• Fast separation of 16 EPA priority pollutants.
  Column EP-100-20-1.5-C18 (1.5mm non-porous ODS,
  Micra Scientific, Inc., Northbrook, IL). Mobile
  phase 70 CH3CN in 30 2mM TRIS. Voltage 55kV.
  Injection 5kV/2s. Detection LIF, ex 257nm, em
  400nm.

16
Gradient Electrochromatography
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Gradient Electrochromatography

• Separation of 16 PAHs
• Column EP-75-26-3-C18. Voltage 20kV for the
  isocratic separations. Injection 5kV/5s.
  Detection LIF, ex 257nm, em 400nm.
• Sample
• 1. naphthalene, 2. acenaphthylene,
• 3. acenaphthene, 4. fluorene,
• 5. phenanthrene, 6. anthracene,
• 7. benzobfluoranthene,
• 8. pyrene,
• 9. benzaanthracene,
• 10. chrysene,
• 11. benzobfluoranthene,
• 12. benzokfluoranthene,
• 13. benzoapyrene,
• 14. dibenza,hanthracene,
• 15. benzoghiperylene, and
• 16. indeno1,2,3-cdpyrene.