Supercritical Fluid Chromatography

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Supercritical Fluid Chromatography

• Theory
• Instrumentation
• Properties of supercritical fluid
• Critical temperature
• Above temperature liquid cannot exist
• Vapor pressure at critical temperature is
  critical pressure
• T and P above critical T and P
• Critical point
• Supercritical fluid

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Supercritical fluid

• Above the critical temperature
• no phase transition regardless of the applied
  pressure
• supercritical fluid is has physical and thermal
  properties that are between those of the pure
  liquid and gas
• fluid density is a strong function of the
  temperature and pressure
• diffusivity much higher a liquid
• readily penetrates porous and fibrous solids
• Low viscosity
• Recovery of analytes
• Return T and P

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Typical Supercritical Solvents
Compound Tcº C Pc atm d
CO2 31.3 72.9 0.96
C2H4 9.9 50.5 ---
N2O 36.5 72.5 0.94
NH3 132.5 112.5 0.40
n-C5 196.6 33.3 0.51
n-C4 152.0 37.5 0.50
CCl2F2 111.8 40.7 1.12
CHF3 25.9 46.9 ----
H2O 374.1 218.3 ----
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Supercritical fluid chromatography

• Combination of gas and liquid
• Permits separation of compounds that are not
  applicable to other methods
• Nonvolatile
• Lack functional groups for detection in liquid
  chromatography

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Supercritical Fluid Extraction

• near the critical point properties change rapidly
  with only slight variations of pressure.
• inexpensive,
• extract the analytes faster
• environmentally friendly
• sample is placed in thimble
• supercritical fluid is pumped through the thimble
  
• extraction of the soluble compounds is allowed to
  take place as the supercritical fluid passes into
  a collection trap through a restricting nozzle
• fluid is vented in the collection trap
• solvent to escapes or is recompressed
• material left behind in the collection trap is
  the product of the extraction
• batch process

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Capillary Electrophoresis

• Separations based on different rate of ion
  migration
• Capillary electrochromatography separates both
  ions and neutral species
• Electroosmotic flow of buffer acts as pump
• Principles
• Applications

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Planar electrophoresis

• porous layer
• 2-10 cm long
• paper
• cellulose acetate
• polymer gel
• soaked in electrolyte buffer
• slow
• difficult to automate

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Capillary Electrophoresis

• narrow (25-75 mm diameter) silica capillary tube
• 40-100 cm long
• filled with electrolyte buffer
• fast
• complex but easy to automate
• quantitative
• small quantities
• nL

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Separation

• Movement of ions function of different parameters
• molecular weight
• charge
• small/highly-charged species migrate rapidly
• pH
• Deprotonation HA?H A-
• ionic strength
• low m
• few counter-ions
• low charge shielding
• high m,
• many counter-ions
• high charge shielding

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Migration rate

• v migration velocity
• meelectrophoretic mobility (cm2/Vs)
• Efield strength (V/cm)
• For capillary
• Vvoltage
• Llength
• Electrophoretic mobility depends on net charge
  and frictional forces
• Size/molecular weight of analyte
• Only ions separated
• Plate height (H) and count (N)
• Function of diffusion and V

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Plates

• Planar electrophoresis
• large cross-sectional area
• short length
• low electrical resistance, high currents
• Sample heating Vmax500 V
• N100-1000 low resolution
• Capillary electrophoresis
• small cross-sectional area
• long length
• high resistance
• low currents
• Vmax20-100 kV
• N100,000-10,000,000 high resolution
• As comparison, HPLC N1,000-20,000

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Zone Broadening

• Single phase (mobile phase) - no partitioning
• three zone broadening phenomena
• longitudinal diffusion
• transport to/from stationary phase
• multipath
• planar
• no stationary phase
• capillary
• no stationary phase or multipath

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Transport

• ions migrating in electric field
• cations to cathode (-ve)
• anions to anode (ve)
• Electroosmosis movement in one direction
• anode (ve) to cathode (-ve)
• Components
• Analyte dissolved in background electrolyte and
  pH buffer
• Silica capillary wall coated with silanol (Si-OH)
  and Si-O-
• Wall attracts cations - double-layer forms
• Cations move towards cathode and sweep fluid in
  one direction
• Electroosmotic flow proportional to V
• usually greater than electrophoretic flow

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Bulk flow properties
hydrodynamic
ion
buffer
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Techniques

• Electropherogram
• migration time analogous to retention time in
  chromatography
• Isoelectric focusing
• Gradient
• No net migration
• pH gradient with weak acid

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Techniques