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 Chromatography

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Supercritical fluid chromatography

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


Supercritical fluid

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


Supercritical fluid chromatography

Typical Supercritical Solvents


Supercritical fluid chromatography1

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


Supercritical fluid extraction

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


Capillary electrophoresis

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


Planar electrophoresis

Planar electrophoresis

  • porous layer

  • 2-10 cm long

    • paper

    • cellulose acetate

    • polymer gel

      • soaked in electrolyte buffer

  • slow

  • difficult to automate


Capillary electrophoresis1

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


Separation

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


Migration rate

Migration rate

  • v= migration velocity

  • me=electrophoretic mobility (cm2/Vs)

  • E=field strength (V/cm)

  • For capillary

    • V=voltage

    • L=length

  • 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


Plates

Plates

  • Planar electrophoresis

    • large cross-sectional area

    • short length

    • low electrical resistance, high currents

    • Sample heating Vmax=500 V

    • N=100-1000 low resolution

  • Capillary electrophoresis

    • small cross-sectional area

    • long length

  • high resistance

  • low currents

    • Vmax=20-100 kV

  • N=100,000-10,000,000 high resolution

    • As comparison, HPLC N=1,000-20,000


Zone broadening

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


Transport

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


Bulk flow properties

Bulk flow properties

hydrodynamic

ion

buffer


Techniques

Techniques

  • Electropherogram

    • migration time analogous to retention time in chromatography

  • Isoelectric focusing

    • Gradient

      • No net migration

    • pH gradient with weak acid


Techniques1

Techniques


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