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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]

Wilkes University -CHM 342

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

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

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
R determined experimentally with static light scattering as well as with
g
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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341

SEC Illustrated

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341

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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341

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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341

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

analyte electrophoretic migration velocity (p)


toward the electrode of opposite charge is:
p = pE

p = 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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341

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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341

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

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341

The Instrumental Requirements

Capillary Electrophoresis

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341

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 microHPLC column

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341

Electrochromatography

Wilkes University - CHM


341

Electrochromatography

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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341

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. 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