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Electrochromatography - A Hybrid Separation Technique. Gel Filtration Chromatography + Capillary Electrophoresis = Electrochromatography [info shamelessly taken from Wikipedia and http://www.unimicrotech.com/products_CEC_instrument.htm]. The Idea.

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electrochromatography a hybrid separation technique

Electrochromatography - A Hybrid Separation Technique

Gel Filtration Chromatography + Capillary Electrophoresis = Electrochromatography

[info shamelessly taken from Wikipedia

and

http://www.unimicrotech.com/products_CEC_instrument.htm]

Wilkes University -CHM 342

the idea
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

Wilkes University - CHM 341

the basics gel filtration or permeation
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

Wilkes University - CHM 341

the basics hydrodynamic volume
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.

Wilkes University - CHM 341

sec illustrated
SEC – Illustrated

Wilkes University - CHM 341

gel filtration or permeation inst
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)

Wilkes University - CHM 341

standard gel electrophoresis
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

Wilkes University - CHM 341

the basics cap electrophoresis
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

Wilkes University - CHM 341

the basics electrophoretic mobility
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 (η) of the medium
    • r is the Stokes radius of the analyte
      • D is the diffusion coefficient.

Wilkes University - CHM 341

the basics electroosmotic flow
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

Wilkes University - CHM 341

the basics electroosmotic flow11
The Basics – electroosmotic flow
  • electroosmotic flow (EOF) of buffer is directed toward the cathode (-)
  • the electroosmotic flow of buffer > 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

Wilkes University - CHM 341

the instrumental requirements
The Instrumental Requirements
  • Capillary Electrophoresis

Wilkes University - CHM 341

electrochromatography
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

Wilkes University - CHM 341

electrochromatography14
Electrochromatography

Wilkes University - CHM 341

electrochromatography15
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.

Wilkes University - CHM 341

gradient electrochromatography
Gradient Electrochromatography

Wilkes University - CHM 341

gradient electrochromatography17
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. benzo[b]fluoranthene,

8. pyrene,

9. benz[a]anthracene,

10. chrysene,

11. benzo[b]fluoranthene,

12. benzo[k]fluoranthene,

13. benzo[a]pyrene,

14. dibenz[a,h]anthracene,

15. benzo[ghi]perylene, and

16. indeno[1,2,3-cd]pyrene.

Wilkes University - CHM 341