1. 19-1 Supercritical Fluid Chromatography Theory Instrumentation Properties of supercritical fluid §Critical temperature àAbove temperature liquid cannot exist àVapor pressure at critical temperature is critical pressure §T and P above critical T and P àCritical point àSupercritical fluid

2. 19-2 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 àreadily penetrates porous and fibrous solids §Low viscosity §Recovery of analytes àReturn T and P

3. 19-3 Typical Supercritical Solvents CompoundT c º CP c atmd*d* CO 2 31.372.90.96 C2H4C2H4 9.950.5--- N2ON2O36.572.50.94 NH 3 132.5112.50.40 n-C 5 196.633.30.51 n-C 4 152.037.50.50 CCl 2 F 2 111.840.71.12 CHF 3 25.946.9---- H2OH2O374.1218.3----

4. 19-4 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. 19-5 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 àsolvent to escapes or is recompressed material left behind in the collection trap is the product of the extraction §batch process

6. 19-6 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. 19-7 Planar electrophoresis porous layer 2-10 cm long §paper §cellulose acetate §polymer gel àsoaked in electrolyte buffer slow difficult to automate

8. 19-8 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. 19-9 Separation Movement of ions function of different parameters §molecular weight §charge àsmall/highly-charged species migrate rapidly §pH àDeprotonation HA  H+ + A- §ionic strength  low  àfew counter-ions àlow charge shielding  high , àmany counter-ions àhigh charge shielding

10. 19-10 Migration rate v= migration velocity  e =electrophoretic mobility (cm 2 /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. 19-11 Plates Planar electrophoresis §large cross-sectional area §short length §low electrical resistance, high currents §Sample heating V max =500 V §N=100-1000 low resolution Capillary electrophoresis §small cross-sectional area §long length high resistance low currents §V max =20-100 kV N=100,000-10,000,000 high resolution §As comparison, HPLC N=1,000-20,000

12. 19-12 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. 19-13 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. 19-14 Bulk flow properties hydrodynamic ion buffer

15. 19-15 Techniques Electropherogram §migration time analogous to retention time in chromatography Isoelectric focusing §Gradient àNo net migration §pH gradient with weak acid

16. 19-16 Techniques