Transcript Electrochromatography

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]
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The Idea
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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
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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
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Size exclusion chromatography (SEC)
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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)
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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
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The Basics – Hydrodynamic Volume
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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
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Gel Filtration or Permeation – Inst.
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HPLC type setup
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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
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Separation uses a “gel" as the stationary
phase – it is often a crosslinked polymer
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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.
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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
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Capillary electrophoresis (CE), also known as
capillary zone electrophoresis (CZE)
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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
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analyte electrophoretic migration velocity (up)
toward the electrode of opposite charge is:
up = μpE
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μp = electrophoretic mobility
E is the electric field strength
electrophoretic mobility at a given pH
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z is the net charge of the analyte
the viscosity (η) of the medium
r is the Stokes radius of the analyte
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D is the diffusion coefficient.
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The Basics – electroosmotic flow
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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
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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
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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
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small multiply charged cations migrate quickly and small multiply charged
anions are retained strongly
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The Instrumental Requirements
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Capillary Electrophoresis
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Electrochromatography
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high efficiency of CE is
combined with the high
selectivity of micro-HPLC
hybrid technique known as
capillary
electrochromatography
(CEC).
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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 microHPLC column
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Electrochromatography
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Electrochromatography
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Fast separation of 16 EPA priority pollutants. Column: EP-100-201.5-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.
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Gradient Electrochromatography
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Gradient Electrochromatography
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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.
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