SFC and SF Extraction

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

18. 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

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

20. 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

21. 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

22. 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

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