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

v = vep + veo

+

-

N

5 = 2 + 3

3 = 0 + 3

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 0.1 M NaCl

+ -

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

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

Mobilizing the bands

UV detector

20 kV Power Supply

-+

flow

Fused silica Capillary50 m ID

pH = 12

Lagging buffur Leading buffer