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 –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, 10 6

Experimental set-up UV detector 20 kV Power Supply ions - ions EO Electrolyte buffer Small sample plug Fused silica Capillary 50  m ID

Mobility Combination of electrophoritic flow and electrosmotic flow v = v ep + v eo v ep =  E V eo is governed by the pH and ionic strength of buffer

v = v ep + v eo + - N 5 = = = 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 Capillary 50  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 Capillary 50  m ID pH = 12

Forming the bands UV detector 20 kV Power Supply - + H + ions OH - ions pH = 2 Sample and ampholytes Fused silica Capillary 50  m ID pH = 12 pI = 4.1pI = 8.3

Mobilizing the bands UV detector 20 kV Power Supply - + H + ions OH - ions pH = 2 Sample and ampholytes Fused silica Capillary 50  m ID pH = 12 pI = 4.1pI = 8.3 Add NaCl Cl - 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 Capillary 50  m ID pH = 12 Lagging buffur Leading buffer