Supercritical Fluid Chromatography

1

• Supercritical Fluid Chromatography
• SFC is a hybrid of HPLC and GC
• Uses HPLC like equipment
• Detector is usually a flame ionization detector,
  although mass selective, and spectrophotmetric
  detectors can be used
• Useful for compounds that
• Are non volatile or thermally unstable and
• Cannot be used with spectophotometric or
  electrochemical detectors
• Properties of supercritical fluids

2

• Supercritical Fluid Chromatography
• Instrumentation
• Similar to HPLC as pressures are like that of
  HPLC
• Need an oven like GC to keep temperature above
  critical temperature of mobile phase
• A restrictor is required at the end of the column
  to maintain the column pressure above that of
  the critical pressure of the mobile phase
• Instead of temperature programming used in GC,
  pressure programming, which increases super
  critical fluid density, is used to modify the
  solvent power of the mobile phase to control
  retention times
• Columns
• Wall coated open tubular columns made from fused
  silica
• Usually chemically bonded liquid stationary phase
• 0.05-0.10 mm ID
• Film thickness 0.05-1.0 mm
• 10-60 m long
• Packed HPLC columns can also be used

3

• Supercritical Fluid Chromatography
• Mobile phase used most is CO2 or CO2 with some
  dissolved methanol which increases the
  solubility of polar compounds
• Nontoxic
• Low TC and PC
• Easily volatilized at the end of the separation,
  leaving solute analytes in the gas phase for
  flame ionization detection
• The FID does not respond to CO2, whereas the mass
  spec does
• Comparison to HPLC and GC based on the fact that
  supercritical fluids have properties
  intermediate between gases and liquids
• Faster elutions than HPLC because the viscosity
  of mobile phase less than liquids allowing for
  higher flow rates
• Lower band broadening than HPLC but greater than
  GC because the diffusion coefficient of solutes
  in supercritical fluids is intermediate between
  that found for gases and liquids
• The overall result is that resolution is greater
  than HPLC but less than GC
• Speed of analyses is greater than HPLC but less
  than GC
• Some figures of merit by example
• See Fig. 27-3 in Skoog, et al.
• At m 0.6 cm/s, SFC gives HETP of 0.013 mm, HPLC
  gives 0.039 mm
• This means zone broadening is reduced by
• HETPminimum is 0.15 cm/s for HPLC, 0.6 cm/s for
  SFC
• This means the same resolution can be obtained in
  one-forth the time
• See Fig. 27-4 in Skoog, et al.

4

• Supercritical Fluid Chromatography
• One disadvantage of SFC is that selectivity
  cannot be modified in the same way as with HPLC
  by using gradient elution. This is important for
  large, nonvolatile molecules
• Applications see Figs. 27-6, 27-7, 27-8 in
  Skoog, et al.
• Capillary Zone Electrophoresis
• Electrophoresis is a separation that is produced
  by differential migration of species in an
  electric field
• In pure electrophoresis, this is not a
  chromatographic separation because no
  partitioning of dissolved analytes occurs between
  a mobile and stationary phase
• It is generally applied to ionic substances,
  although some new techniques allow separation of
  neutral molecules by combining electrophoresis
  with chromatography

5
Capillary Zone Electrophoresis Electroosmotic
flow causes the solvent to move from the vessel
with the negative electrode to the vessel with
the positive electrode
Negative electrode

• Positive adsorbed ions are attracted to the
  negative electrode
• Viscous drag of the mobile positive ions in
  solvent pulls the solvent along with the ions
• The solvent front has a very flat profile unlike
  that of a chromatographic mobile phase
• Separation occurs because of differences in the
  mobilities analyte solute molecules
• Charge
• Size
• Shape
• Solvent properties such as pH, dielectric
  constant, concentration of buffer, ionic
  strength
• Positive analyte ions move faster than the rate
  of electroosmotic flow
• Negative analyte ions move slower that
  electroosmotic flow rate

6

• Capillary Zone Electrophoresis
• Sample injection can be accomplished by siphoning
  some of the analyte solution into the end of the
  capillary or by electroosmotic flow
• Applications see Fig. 27-11 in Skoog, et al.
  showing the separation of some proteins
• Micellar electrokinetic capillary chromatography
  (MECC) involves suspending micelles in the
  mobile phase which allows partitioning of analyte
  solute molecules - including neutrals - between
  the moving mobile phase and the moving micelles
• This can be viewed as a simultaneous
  electrophoretic and chromatographic separation