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_instrument.htm] Wilkes University -CHM 342

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 Wilkes University - CHM 341

3. 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

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. Wilkes University - CHM 341

5. SEC – Illustrated Wilkes University - CHM 341

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) Wilkes University - CHM 341

7. 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

8. 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

9. 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

10. 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

11. 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

12. The Instrumental Requirements • Capillary Electrophoresis Wilkes University - CHM 341

13. 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

14. Electrochromatography Wilkes University - CHM 341

15. 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

16. Gradient Electrochromatography Wilkes University - CHM 341

17. 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