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
19-1
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
19-2
Typical Supercritical Solvents
Compound
Tcº C
Pc atm
d*
CO2
C2H4
N2O
NH3
31.3
9.9
36.5
132.5
72.9
50.5
72.5
112.5
0.96
--0.94
0.40
n-C5
n-C4
CCl2F2
196.6
152.0
111.8
33.3
37.5
40.7
0.51
0.50
1.12
CHF3
H2O
25.9
374.1
46.9
218.3
------19-3
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
19-4
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
19-5
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
19-6
Planar electrophoresis
• porous layer
• 2-10 cm long
 paper
 cellulose acetate
 polymer gel
 soaked in
electrolyte
buffer
• slow
• difficult to automate
19-7
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
19-8
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
19-9
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)
19-10
 Function of diffusion and V
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
19-11
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
19-12
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 SiO
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
19-13
Bulk flow properties
hydrodynamic
ion buffer
19-14
Techniques
• Electropherogram
 migration time
analogous to
retention time in
chromatography
• Isoelectric focusing
 Gradient
 No net migration
 pH gradient with
weak acid
19-15
Techniques
19-16