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SFC and SF Extraction

SFC and SF Extraction. Intermediate between HPLC and GC SF are substances above their critical pressure and temperature (critical point SFs has great solvating power and high diffusivity (CO 2 most common, CT = 31  C, CP = 73 atm) Can solvate non-volatiles Can flow at high linear flow rates

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SFC and SF Extraction

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  1. SFC and SF Extraction • Intermediate between HPLC and GC • SF are substances above their critical pressure and temperature (critical point • SFs has great solvating power and high diffusivity (CO2 most common, CT = 31C, CP = 73 atm) • Can solvate non-volatiles • Can flow at high linear flow rates • Can use longer columns • Can connect to GC or HPLC detectors • Pressure/Temp gradients, mobile phase actively participates in separation; MeOH additives

  2. Capillary Electrophoresis • Small open tubular capillary • High voltage • Electrolyte • Small sample plug • Electrophoretic mobility • m = (q/f)(E) • detector

  3. Why cap electrophoresis? • Separation of ions • High separation efficiency • No stationary phase • Plug profile • Only longitudinal diffusion term • Very high plate numbers, 106

  4. Experimental set-up 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector EO + ions - ions Small sample plug Electrolyte buffer

  5. Mobility • Combination of electrophoritic flow and electrosmotic flow • v = vep + veo vep = mE Veo is governed by the pH and ionic strength of buffer

  6. v = vep + veo 5 = 2 + 3 + 3 = 0 + 3 N 1 = -2 + 3 - + N -

  7. Challenges • Need a small sample size (concentrated sample) • Pre-concentrate large sample • stacking • Can not separate neutrals • Add micelles • Pre-concentrate large sample • stacking

  8. Stacking • Fill capillary with buffer of weaker ionic strength, 0.10 NaCl • Add a large plug of sample with higher ionic strength • Create a sandwich by adding weaker buffer • Apply voltage for a brief while • Change leads and apply voltage for a while • Change back and start analysis

  9. Fill with 0.1 M NaCl + -

  10. Fill with sample 0.01 M NaCl 0.1 M NaCl + -

  11. Apply voltage 0.01 M NaCl 0.1 M NaCl + -

  12. Switch Electrodes 0.01 M NaCl 0.1 M NaCl - +

  13. Switch Back and begin separation 0.01 M NaCl 0.1 M NaCl + -

  14. Different Types of CE • Capillary Zone Electrophoresis • Small ions • Capillary isoelectric focusing • Amphoteric compounds • Cap. Gel Electrophoresis • Slab for proteins and DNA • Cooling/sieving mechanism • polyacrylamide • Capillary isotachophoresis • Capillary electrochromatography • Micellar Electrokinetic chromatography

  15. CZE 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector EO + ions - ions Small sample plug Electrolyte buffer

  16. Capallary Gel Electrophoresis • Slab Gel Electrophoresis for proteins and DNA • Cooling/sieving mechanism • Polyacrylamide • Some capillary applications, as well • 2 D Gel Electrophoresis • Separates by size and pI

  17. Capillary isoelectric focusing-CIEF • Separation of amphoteric species – such as a protein • pH gradient established • A protein will move along the gradient until they reach a pH that correspond to its pI, the pH where the average charge is zero • Resolution, 0.2 pI units • Mobilization of the bands

  18. CIEF 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector H+ ions OH- ions Sample and ampholytes pH = 2 pH = 12

  19. Forming the bands 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector H+ ions pI = 4.1 pI = 8.3 OH- ions Sample and ampholytes pH = 2 pH = 12

  20. Mobilizing the bands 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector H+ ions pI = 4.1 pI = 8.3 OH- ions Add NaCl Cl- ions Sample and ampholytes pH = 2 pH = 12

  21. Capillary Isotachophoresis • Sandwich sample between a leading and a lagging buffer • Leading buffer is faster than each of the analytes • Lagging buffer is slower than each of the analytes • Analytes form bands between buffers • Once band form they whole solution in the capillary moves at a constant velocity

  22. Mobilizing the bands 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector flow Leading buffer Lagging buffur pH = 12

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