Supercritical Fluid Chromatography

1
Supercritical Fluid Chromatography
Introduction Supercritical Fluid
Chromatography (SFC) is a hybrid of gas and
liquid chromatography that combines some of the
best features of both Supercritical Fluid exist
at temperatures and pressures above its critical
temperature and pressure and have densities,
viscosities and other properties that are
intermediates between those of the substance in
its gaseous and liquid state.
Gas (STP) Supercritical Fluid Liquid
Density (g/cm3) (0.6-2) x 10-3 0.2-0.5 0.6-2
Diffusion coefficient (cm2/s) (1-4)x10-1 10-3-10-4 (0.2-2)x10-5
Viscosity (g cm-1 s-1) (1-3)x10-4 (1-3)x10-4 (0.2-3)x10-2
Critical temperature temperature above which a
distinct liquid phase does not exist regardless
of pressure
2
Important Properties of Supercritical fluids
remarkable ability to dissolve large,
non-volatile molecules e.g., supercritical
CO2 can dissolve n-alkanes containing over 30
carbon atoms related to their high
densities dissolved analytes are easily
recovered equilibrate with atmosphere at
relatively low temperatures e.g., analyte
in supercritical CO2 can be recovered by reducing
the pressure and allowing the CO2 to
evaporate no need for organic solvents
environmentally friendly inexpensive,
innocuous and non-toxic higher diffusion
coefficients and lower viscosities
relative to liquids faster and higher
resolution separations
3
Advantages of SFC compared to LC and GC - SFC
can separate compounds that are not conveniently
handled by GC or LC. non-volatile or
thermally labile and contain no
functional group that makes possible detection in
LC using spectroscopic or
electrochemical techniques lt up to 25 of all
separation problems fall into this category lt
examples include polymers, fossil fuels,
pesticides, foods, drugs, etc. -
Separations are faster then LC - Run at
lower temperature than GC - Beneficial in
industrial scale purification
Reduction in peak width (p3)
Faster elution
Reduction in elution time (4x)
4
Instrumentation Instrumentation for SFC is
very similar to ordinary HPLC equipment since the
temperature pressure requirements for
supercritical fluids fall within the standard
operation
Two Major Differences thermostated column
oven requires precise temperature control of
mobile phase (typically supercritical CO2)
restrictor or back-pressure device required
to maintain desired pressure in column
pressure change to convert from supercritical
fluid to a gas for transfer to detector
5
Effects of Pressure Pressure increases results
in reduced elution time ? - increase in
density of mobile phase - effects retention or
capacity factor (k) - pressure changes ?
analogous to gradient elution in LC and GC
6
Example 15 What properties of a supercritical
fluid are important in chromatography?
7
Electrophoresis
Introduction Electrophoresis is a technique
in which solutes are separated by their different
rates of travel through an electric field. -
commonly used in biological analysis,
particularly in the separations of
proteins, peptides and nucleic acids
Similar to
chromatography, both involve separation by
different rates of travel through
the system, but electrophoresis ?
different rates of travel are produced by an
electric field chromatography ? different rates
of travel due to chemical interactions
between the solutes and a stationary or
mobile phase
8
Theory The migration rate of solutes in an
electric field will depend on the size and charge
of the solute, as well as other
factors Retention The degree of retention,
or rate of travel, of a solute in an
electrophoretic system is given
by v m E where E electric
field strength v Velocity of the solute in
the electric field m Electrophoretic
mobility of the solute The value of m is a
constant for a given solute under a particular
set of experimental conditions and depends on
the following ionic charge on the
analyte inversely proportional to
frictional retarding factors - size of
analyte - shape of analyte - viscosity of
medium Note charge-to-size ratio of analyte is
an important factor in its mobility ? small/high
charged molecules migrate faster
large/low charged molecules migrate slower
9
Efficiency Electrophoresis may be performed in
an open-tubular system (capillary) or in a
packed-bed (slab)
Packed-bed (slab) system
Capillary system
10
Joule Heating uneven heating in the system
by the electric field causes different points
in the system to have different temperatures
non-uniform mixing of solute and solvent
gives rise to peak broadening in
electrophoresis decrease band-broadening
caused by Joule heating use of packed-bed
systems (or stabilizers) ? prevents mixing of
solvent from different regions of the
system use of more efficient cooling ?
prevents the formation of thermal gradients
Result of Joule Heating
11
Zone Electrophoresis Involves applying narrow
bands of sample to the electrophoretic system -
common type of electrophoresis - buffer
composition is constant throughout system -
solutes can be totally separated in a reasonable
amount of time into separate zones based on
mobility - zones are separated by regions of
buffer
Visualize bands with UV light
Apply voltage across gel
Load samples into each well
12
Zone Electrophoresis
Packed-bed (slab) much work in zone
electrophoresis uses packed-bed instead of open
tubular supports avoids band-broadening from
Joule heating and unstable density gradients
packed beds stabilize solvent in the system
prevents mixing between different regions
common supports cellulose acetate, paper,
agarose and polyacrylamide gels
band-broadening caused by diffusion around
particles of the packed bed porous matrix may
also result in molecules being separated
according to size in addition to
electrophoretic mobility ? SEC
13
Capillary electrophoresis (CE) another type
of zone electrophoresis use of narrow open
tubular supports or capillaries typically
25-75 mm I.D. narrow bore capillaries allows
efficient removal of Joule heat from the
system decrease band-broadening allows
stronger electric fields to be used lt faster
migration of solutes ? decreasing analysis
time easy to automate very small
sample sizes (nl) no particles ? no multiple
paths many types of detectors can be used in
capillary electrophoresis Fluorescence or
UV/Vis absorbance are commonly used
14
Capillary electrophoresis (CE)
15
Because of the extreme efficiency and narrow
peaks produced by CE, it is an area of intense
research - especially in areas of biotechnology
and biochemical separations - very small sample
requirements - useful in separating and
studying the ionic solutes present in single
cells - screening of large chemical libraries
for drug discovery - genome sequencing
CE separation of DNA mixtures
16
Electroosmotic flow all solutes, regardless of
charge, will migrate towards the negative
electrode of the system - feature of capillary
electrophoresis - produced by the presence of
negative charge on the surface of the
capillary negative charge is
produced by ionization of silanol groups on the
capillarys surface occurs at pH gt
4-5 net positive charge must exist in
solution to counter negative charge of
capillarys surface net positive charge of
solution causes net migration toward the
negative electrode - ALL solutes eventually
pass detector, regardless of charge
17
Electroosmotic flow all solutes, regardless of
charge, will migrate towards the negative
electrode of the system - flow
profile is flat ? no peak broadening
pressure-driven flow in LC causes parabolic
profile caused by boundary effects and
inertia
Direction velocity due to potential
Overall direction velocity
u(electrophoretic) u(electrophoretic)
u(electroosmotic)
Direction of Electroosmotic flow
Anode ()
Cathode (-)
18
Isoelectric focusing pH affects charge which
affects migration HA H A- Ka
HA-/HA For a protein composed of amino
acids


Low pH (acidic)
pI
High pH (basic)
isoelectric point (pI) - pH at which net charge
on molecule is zero If plate has pH
gradient, protein migrates until local pH pI
19
Example 16 Three large proteins are ionized at
the pH at which a capillary electrophoresis
separation is carried out. If the ions are
designated A2, B and C3, predict
the order of the elution?