Supercritical Fluid Chromatography

1. Supercritical Fluid Chromatography • Theory • Instrumentation • Properties of supercritical fluid • Critical temperature • Above temperature liquid cannot exist • Vapor pressure at critical temperature is critical pressure • T and P above critical T and P • Critical point • Supercritical fluid

2. Supercritical fluid • Above the critical temperature • no phase transition regardless of the applied pressure • supercritical fluid is has physical and thermal properties that are between those of the pure liquid and gas • fluid density is a strong function of the temperature and pressure • diffusivity much higher a liquid • readily penetrates porous and fibrous solids • Low viscosity • Recovery of analytes • Return T and P

3. Typical Supercritical Solvents

4. Supercritical fluid chromatography • Combination of gas and liquid • Permits separation of compounds that are not applicable to other methods • Nonvolatile • Lack functional groups for detection in liquid chromatography

5. Supercritical Fluid Extraction • near the critical point properties change rapidly with only slight variations of pressure. • inexpensive, • extract the analytes faster • environmentally friendly • sample is placed in thimble • supercritical fluid is pumped through the thimble • extraction of the soluble compounds is allowed to take place as the supercritical fluid passes into a collection trap through a restricting nozzle • fluid is vented in the collection trap • solvent to escapes or is recompressed • material left behind in the collection trap is the product of the extraction • batch process

6. Capillary Electrophoresis • Separations based on different rate of ion migration • Capillary electrochromatography separates both ions and neutral species • Electroosmotic flow of buffer acts as pump • Principles • Applications

7. Planar electrophoresis • porous layer • 2-10 cm long • paper • cellulose acetate • polymer gel • soaked in electrolyte buffer • slow • difficult to automate

8. Capillary Electrophoresis • narrow (25-75 mm diameter) silica capillary tube • 40-100 cm long • filled with electrolyte buffer • fast • complex but easy to automate • quantitative • small quantities • nL

9. Separation • Movement of ions function of different parameters • molecular weight • charge • small/highly-charged species migrate rapidly • pH • Deprotonation HAH+ + A- • ionic strength • low m • few counter-ions • low charge shielding • high m, • many counter-ions • high charge shielding

10. Migration rate • v= migration velocity • me=electrophoretic mobility (cm2/Vs) • E=field strength (V/cm) • For capillary • V=voltage • L=length • Electrophoretic mobility depends on net charge and frictional forces • Size/molecular weight of analyte • Only ions separated • Plate height (H) and count (N) • Function of diffusion and V

11. Plates • Planar electrophoresis • large cross-sectional area • short length • low electrical resistance, high currents • Sample heating Vmax=500 V • N=100-1000 low resolution • Capillary electrophoresis • small cross-sectional area • long length • high resistance • low currents • Vmax=20-100 kV • N=100,000-10,000,000 high resolution • As comparison, HPLC N=1,000-20,000

12. Zone Broadening • Single phase (mobile phase) - no partitioning • three zone broadening phenomena • longitudinal diffusion • transport to/from stationary phase • multipath • planar • no stationary phase • capillary • no stationary phase or multipath

13. Transport • ions migrating in electric field • cations to cathode (-ve) • anions to anode (+ve) • Electroosmosis movement in one direction • anode (+ve) to cathode (-ve) • Components • Analyte dissolved in background electrolyte and pH buffer • Silica capillary wall coated with silanol (Si-OH) and Si-O- • Wall attracts cations - double-layer forms • Cations move towards cathode and sweep fluid in one direction • Electroosmotic flow proportional to V • usually greater than electrophoretic flow

14. Bulk flow properties hydrodynamic ion buffer

15. Techniques • Electropherogram • migration time analogous to retention time in chromatography • Isoelectric focusing • Gradient • No net migration • pH gradient with weak acid

16. Techniques