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

19-7

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 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

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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

19-13

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

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