19-1 supercritical fluid chromatography theory instrumentation properties of supercritical fluid...
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19-1
Supercritical Fluid Chromatography
• Theory• Instrumentation
• Properties of supercritical fluid Critical temperature
Above temperature liquid cannot existVapor pressure at critical temperature is
critical pressure T and P above critical T and P
Critical pointSupercritical fluid
19-2
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
19-3
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
19-5
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
19-6
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
19-8
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
19-9
Separation
• Movement of ions function of different parameters molecular weight charge
small/highly-charged species migrate rapidly pH
Deprotonation HAH+ + A- ionic strength
low few counter-ions low charge shielding
high , many counter-ions high charge shielding
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Migration rate
• v= migration velocity e=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
19-11
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
19-12
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
19-14
Bulk flow properties
hydrodynamic
ion buffer
19-15
Techniques• Electropherogram
migration time analogous to retention time in chromatography
• Isoelectric focusing Gradient
No net migration pH gradient with
weak acid
19-16
Techniques