wilkes university -chm 342 electrochromatography - a hybrid separation technique gel filtration...

Wilkes University -CHM 342

Electrochromatography - A Hybrid Separation Technique

Gel Filtration Chromatography + Capillary Electrophoresis = Electrochromatography

[info shamelessly taken from Wikipediaand

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

Wilkes University - CHM 341

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


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


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 beunderstood 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.


SEC – Illustrated


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)


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


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


The Basics – Electrophoretic Mobility

analyte electrophoretic migration velocity (p) toward the electrode of opposite charge is:

p = 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.


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


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


The Instrumental Requirements

Capillary Electrophoresis


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


Electrochromatography


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.


Gradient Electrochromatography


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 342 electrochromatography - a hybrid separation technique gel filtration...

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