sfc and sf extraction intermediate between hplc and gc sf are substances above their critical...
Post on 20-Dec-2015
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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 = (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
UV detector
20 kV Power Supply
-+
+ ions
- ions
EO
Electrolyte buffer
Small sample plug
Fused silica Capillary50 m ID
Mobility
• Combination of electrophoritic flow and electrosmotic flow
• v = vep + veo
vep = E
Veo is governed by the pH and ionic strength of buffer
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
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
UV detector
20 kV Power Supply
-+
+ ions
- ions
EO
Electrolyte buffer
Small sample plug
Fused silica Capillary50 m ID
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
UV detector
20 kV Power Supply
-+
H+ ions
OH- ions
pH = 2
Sample and ampholytes
Fused silica Capillary50 m ID
pH = 12
Forming the bands
UV detector
20 kV Power Supply
-+
H+ ions
OH- ions
pH = 2
Sample and ampholytes
Fused silica Capillary50 m ID
pH = 12
pI = 4.1 pI = 8.3
Mobilizing the bands
UV detector
20 kV Power Supply
-+
H+ ions
OH- ions
pH = 2
Sample and ampholytes
Fused silica Capillary50 m ID
pH = 12
pI = 4.1 pI = 8.3
Add NaClCl- ions
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