SFC and SF Extraction

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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 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

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Capillary Electrophoresis

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

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Why cap electrophoresis?

• Separation of ions
• High separation efficiency
• No stationary phase
• Plug profile
• Only longitudinal diffusion term
• Very high plate numbers, 106

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Experimental set-up
20 kV Power Supply
-

Fused silica Capillary 50 mm ID
UV detector
EO
ions
- ions
Small sample plug
Electrolyte buffer
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Mobility

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

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v vep veo
5 2 3

3 0 3
N
1 -2 3
-

N
-
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Challenges

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

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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

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Fill with 0.1 M NaCl

-
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Fill with sample
0.01 M NaCl
0.1 M NaCl

-
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Apply voltage
0.01 M NaCl
0.1 M NaCl

-
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Switch Electrodes
0.01 M NaCl
0.1 M NaCl
-

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Switch Back and begin separation
0.01 M NaCl
0.1 M NaCl

-
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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

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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

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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

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CIEF
20 kV Power Supply
-

Fused silica Capillary 50 mm ID
UV detector
H ions
OH- ions
Sample and ampholytes
pH 2
pH 12
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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
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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
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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

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Mobilizing the bands
20 kV Power Supply
-

Fused silica Capillary 50 mm ID
UV detector
flow
Leading buffer
Lagging buffur
pH 12