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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 • 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 = 31C, 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
Capillary Electrophoresis • Small open tubular capillary • High voltage • Electrolyte • Small sample plug • Electrophoretic mobility • m = (q/f)(E) • detector
Why cap electrophoresis? • Separation of ions • High separation efficiency • No stationary phase • Plug profile • Only longitudinal diffusion term • Very high plate numbers, 106
Experimental set-up 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector EO + ions - ions Small sample plug Electrolyte buffer
Mobility • Combination of electrophoritic flow and electrosmotic flow • v = vep + veo vep = mE Veo is governed by the pH and ionic strength of buffer
v = vep + veo 5 = 2 + 3 + 3 = 0 + 3 N 1 = -2 + 3 - + N -
Challenges • Need a small sample size (concentrated sample) • Pre-concentrate large sample • stacking • Can not separate neutrals • Add micelles • Pre-concentrate large sample • stacking
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
Fill with sample 0.01 M NaCl 0.1 M NaCl + -
Apply voltage 0.01 M NaCl 0.1 M NaCl + -
Switch Electrodes 0.01 M NaCl 0.1 M NaCl - +
Switch Back and begin separation 0.01 M NaCl 0.1 M NaCl + -
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
CZE 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector EO + ions - ions Small sample plug Electrolyte buffer
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
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
CIEF 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector H+ ions OH- ions Sample and ampholytes pH = 2 pH = 12
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
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
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
Mobilizing the bands 20 kV Power Supply - + Fused silica Capillary 50 mm ID UV detector flow Leading buffer Lagging buffur pH = 12