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TOPIC 4
 Course Learning Outcomes
Able to :
1. Explain the fundamental concepts & theories of
separation techniques in SFC & SFE.

2. Sketch, label the schematic diagrams & discuss

the function of each component in SFC & SFE.

3. Identify the strength & limitations of SFC & SFE

technique.

4. Suggest and justify the most suitable & efficient

separation technique to be employed for an
analysis.
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 What is supercritical fluid?
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™ Critical temperature (Tc) for any substance is a

temperature above which it can no longer exist as a
liquid, no matter how much pressure is applied.

™ Critical pressure (Pc) is a pressure above which the

substance can no longer exist as a gas no matter how
high the temperature is raised.
™ A supercritical fluid is
any substance that is CRITICAL
above its Tc & Pc POINT

[critical point].

™ In other words, when a substance is above Tc &

Pc, it exists in a single phase which is neither
liquid nor gas; this is a supercritical fluid.

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Example :

™ CO2 usually behaves as a gas in air at STP or as a

solid called dry ice when frozen.

™ If the temperature & pressure are both increased

from STP to be at or above the critical point for
CO2, it can adopt properties midway between a gas
& a liquid.

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™ More specifically, it behaves as a supercritical
fluid above its Tc (31.1 °C) & Pc (72.9 atm/7.39
MPa), expanding to fill its container like a gas but
with a density like that of a liquid.

™ Above 304.2 K (31.2 oC) & 72.8 atm, CO2

behaves as a supercritical fluid & shows
properties of both a liquid & a gas.

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™ Supercritical fluid has the unique ability :

9 Diffuse through solids like a gas.

9 Dissolve materials like a liquid.

9 It can readily change in density upon minor

changes in temperature or pressure.

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™ Characteristics of supercritical fluid :

9 They have a combination of vapor & liquid

properties.

9 They have densities higher than gas & lower

than liquid & viscosities less than liquids.

9 Their diffusivities are gas-like.

9 They transfer mass very rapidly.

9 They are compressible and homogeneous.

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 Supercritical Fluid Chromatography
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9 The sample is carried through a separating column

by a supercritical fluid where the mixture is divided
into unique bands based on the amount of
interaction between the individual analytes & the
stationary phase in the column.

9 As these bands leave the column their identities

and quantities are determined by a detector.
™ What differentiates SFC from other
chromatographic techniques (GC & HPLC) is
the use of a supercritical fluid as the mobile
phase.

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™ SFC is a hybrid of GC & LC because when the
mobile phase is below its Tc & above its Pc, it
acts as a liquid, so the technique is LC & when
the mobile phase is above its Tc & below its Pc, it
acts as a gas, so the technique is GC.

™ Thus, SFC combines some of the best features of

LC as well as GC.

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™ Theory of separation in SFC is based on the
density of the supercritical fluid which
corresponds to solvating power.

™ As the pressure in the system is increased, the

supercritical fluid density increases &
correspondingly its solvating power increases.

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™ A small increase in pressure causes a large
increase in the density of the supercritical phase.

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™ Solvation (or dissolution), is the process of attraction
and association of molecules of a solvent with molecules
or ions of a solute.

™ As ions dissolve in a solvent they spread out and become

surrounded by solvent molecules.

A sodium ion solvated

by water molecules

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™ Therefore, as the density of the supercritical fluid
mobile phase is increased,

9 Components most retained in the column can be

eluted faster.

9 This will shorten the elution time.

9 This is similar to temperature programming in

GC or using a solvent gradient in HPLC.

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™ E.g., the density varied during the
chromatography is shown by the dashed curve.

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 SFC advantages over GC & HPLC
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™ Compared with gas, supercritical fluid has high

density (liquid-like) (higher than gas, lower than
liquid), therefore able to dissolve large, non volatile
molecules & increase column loading.

™ Compared with liquid, supercritical fluid has low

viscosities, therefore able to use higher flow rate
for faster analysis.
 Sample problem 1
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Briefly discuss the property of supercritical fluid that

relates to its ability to dissolve & separate non volatile
compounds.
 Sample problem 2
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Which property relates to the ability of supercritical

fluid for faster analysis ?
™ Compared with liquids & gases :

9 The diffusion rates of solutes in supercritical fluids is

about 10X greater than that in liquids (and about
3X less than in gases).

9 This results in a decrease in resistance to mass

transfer in the column & allows for fast
separations (faster than HPLC).

9 Band broadening is greater than in HPLC but less

than in GC.

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™ Compared with HPLC,

9 SFC provides rapid separations without the

use of organic solvents (GREEN
TECHNOLOGY).

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™ Compared with GC,

9 Capillary SFC can provide high resolution

chromatography at much lower
temperatures (CO2 Tc = 31.1 °C). This
allows fast analysis of thermolabile
(thermally unstable) compounds.

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™ In term of instrumentation, SFC is compatible with both
HPLC & GC detectors, thus SFC permits the separation
of :

9 Non-volatile or thermally unstable compounds

which are inapplicable by GC.

9 Compounds contain no functional groups which

cannot be detected by the spectroscopic or
electrochemical techniques employed in HPLC.

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 Sample problem 3
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SFC allows the determination of a group of compounds that

are not conveniently handled by either GC or HPLC. What
are these compounds?
 Instrumentation
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Diagram of SFC instrument

™ The instrumentation of SFC is similar in most
regards to instrumentation for HPLC & GC because
the pressure & temperature required for creating
supercritical fluid.

™ Major modification is the replacement of either the

liquid or gas mobile phase with a supercritical fluid
mobile phase.

™ There are 2 main differences between the 2.

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1. A thermostated oven similar to that of GC, is
required to provide precise temperature control of
the mobile phase.

2. A restrictor or a back pressure device (placed

either after the detector or at the end of the column)
to maintain mobile phase supercritical
throughout the separation, prevent pressure drop
& to convert the eluent from SCF to a gas for
transfer to the detector.

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™ In SFC,

9 The mobile phase is initially pumped as a

liquid/gas & is brought into the supercritical region
by heating it above its supercritical temperature
before it enters the analytical column.

9 It passes through an injection valve where the

sample is introduced into the supercritical stream &
then into the analytical column.

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 Mobile phase
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™ CO2 is the most used mobile phase for SFC.

™ Other substances have served as mobile phases

for SFC, including ethane, butane, nitrous oxide,
diethyl ether, ammonia & THF.
™ Advantages of CO2 :

9 Its low cost, low interference with chromatographic

detectors.

9 Good physical properties (nontoxic, low critical

values, nonflammable).

9 It is an excellent solvent for a variety of non-polar

organic molecules.

9 It permits a wide selection of temperatures &

pressures without exceeding the operating limits of
modern HPLC equipments.
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™ The main disadvantage of CO2 (non polar) is not a
very good solvent for high molecular weight, ionic
& polar analytes.

™ This can be overcome by adding a small portion of a

second fluid called modifier fluid.

™ Modifier fluids are commonly used, especially in

packed column SFC.

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™ Modifier fluid :

9 An organic solvent, which is more polar &

completely miscible with CO2.

9 E.g. alcohol (methanol, ethanol, 1-propanol),

ACN & water.

9 Small concentrations (1-20 mol %) to modify

solvation power of CO2.

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™ The addition of the modifier fluid :

9 Enhances the polarity of CO2, thus improves the

solvating ability of the supercritical fluid.

9 Enhances selectivity of the separation.

9 It can also help improve separation efficiency by

blocking some of the highly active sites on the
stationary phase.

9 For highly retained non polar solutes, modifiers

increase the column efficiency.

9 For polar solutes, they improve retention & efficiency,

both.
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 Sample problem 4
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Describe 2 approaches to improve the solvating power of

CO2.
 Column
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™ 2 types of analytical columns :

1. Packed column (stationary phases such as C18 or

aminopropyl bonded silica).

2. Capillary column (capillary columns are open tubular

columns of narrow I.D. made of fused silica, with the
stationary phase bonded to the wall of the column).
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 Pumps
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™ The type of high pressure pump used in SFC is

determined by the column type.
™ Packed columns - reciprocating pumps
™ Capillary columns - syringe pumps

Reciprocating pumps allow easier mixing of the

mobile phase or introduction of modifier fluids
Syringe pumps provide consistent
pressure for a mobile phase
 Injector
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™ For packed SFC, a typical LC injection valve is used.

™ In capillary SFC, small sample volumes must be

quickly injected into the column & therefore
pneumatically driven valves are used.
 Oven
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™ A thermostated column oven is required for

precise temperature control of the mobile
phase.

™ Conventional GC or LC ovens are generally

used.
 Detectors
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™ SFC is compatible with both HPLC & GC detectors.

™ GC detectors - flame ionization & flame photometric

detectors.

™ LC detectors - refractive index, UV-Vis

spectrophotometric & light scattering detectors have
been employed for SFC.
™ Mass spectrometry are more easily adapted
as detector for SFC than in HPLC.

™ FT-IR can also be used.

The choice of detectors will depend upon the mobile

phase composition, column type, flow rate & ability
to withstand the high pressures of SFC.

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 Sample problem 5
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What is the major advantage of SFC over HPLC in

term of instrumentation ?
Effects of pressure (pressure programming)
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™ Part of the theory of separation in SFC is based on

the density of the supercritical fluid which
corresponds to solvating power.

™ As the pressure in the system is increased, the

density of the CO2 increases & correspondingly its
solvating power increases.
™ Therefore, as the density of the supercritical fluid
mobile phase is increased, components retained in
the column can be made to elute .

™ This effect is similar to temperature programming in

GC or gradient elution in HPLC.

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 Sample problem 6
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Describe the kind of gradient which is used in SFC.

 Sample problem 7
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Discuss the advantage of pressure programming in

SFC.
 Sample problem 8
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Discuss the suitable approach to separate a

sample mixture containing volatile & non volatile
compounds with a wide range of polarity by using
SFC.
 Applications
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In practice, the advantages offered by SFC have not

been sufficient to displace the widely used HPLC and
GC, except in a few cases such as chiral
separations & analysis of high-molecular-weight
hydrocarbons.
 Applications
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™ By now SFC has been applied to wide variety

of materials including :

9 Natural products, drugs, foods, pesticides,

herbicides, surfactants, polymers and
polymer additives, fossils fuels, petroleum,
explosives and propellants.
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TOPIC 1
 Introduction
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™ Extraction
Process of obtaining something from a
mixture or compound by chemical or
physical or mechanical means.

Mechanical separations e.g. filtration of a solid from a

suspension in a liquid, centrifugation, screening etc
 Supercritical fluid (SF)
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™ SF can be used to extract analytes from sample.

™ Advantages of SF :
9 Inexpensive
9 Contaminant free
9 Less cost to dispose effluent than organic solvent.
9 Solvating power similar to organic solvent, but with
higher diffusivities, lower viscosity & surface tension.
™ The solvating power of SF can be adjusted by :

9 Changing the pressure or temperature.

9 Adding modifier (MeOH, 1-10%) to increase

the polarity of SF.

Lower or no surface tension in a SF, as there is no

liquid/gas phase boundary. By changing the pressure
and temperature of the fluid, the properties can be
“tuned” to be more liquid- or more gas-like.

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 Supercritical fluid extraction (SFE)
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™ SFE is the process of separating one component (the

extractant) from another (the matrix) using SF as the
extracting solvent.

™ Extraction is usually from a solid matrix, but can also

be from liquids.

™ CO2 is the most used SF (cheap, inert, non toxic).

 Advantages of SFE
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™ Fast due to greater rate of mass transfer between a

sample matrix & extraction fluid (high diffusion rate & low
viscosity).

¾ The rate of mass transfer between a sample matrix &

an extraction fluid is determined by the rate of diffusion
of a species in the fluid & the viscosity of the fluid. The
greater the diffusion rate & the lower the viscosity, the
greater will be the rate of mass transfer.
™ Hence, compared with solvent extraction (several
hours or days), SFE required 10–60 mins.

™ Does not use large amounts of solvent to extract

samples (environmental friendly).

™ Good for extraction of non-volatile components.

Solvent strength of SF can be varied by changes in
pressure (selectivity).

™ In contrast, solvent strength of organic solvent

extraction is constant regardless of condition.

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™ Recovery of analytes are simple :

9 Extracted material is easily recovered by simply

depressurizing, allowing the SF to return to gas
phase & evaporate leaving no or little solvent
residues.

9 Compared to solvent extraction whereby vaporized

by heating which leads to possibility of
decomposition or loss of volatile analytes.

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 Sample problem 1
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For a SFE, describe its advantages over solvent extraction.

 SFE applications
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™ Analysis of environmental samples (pesticides,

PAHs, PCBs etc).
™ Analysis of food (fat etc.).
™ Biomedical (cholesterol, drug residues etc.)
™ Industrial samples.
Instrumentation
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™ A SF extractor consists of :

9 A tank of the mobile phase, usually CO2,

9 A pump to pressurize the gas (e.g. syringe pump

pressure at least 400 atm & flow rate at least a few
mL/min,

9 An oven containing the extraction vessel/cell

(capacity of a few mL),

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9 A flow restrictor to maintain a high pressure in the
extraction line (depressurizes the fluid & transfer it
into a collection device),

9 A trapping vessel/sample collection/collector - either

a vial with solvent (solvent trapping) or solid sorbent
trap (sorbent trapping).

9 On-line interface (after flow restrictor) – connected

directly to a chromatographic system.

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 Procedure for SFE
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™ The system must contain a pump for the CO2, a

pressure cell to contain the sample, a means of
maintaining pressure in the system and a
collecting vessel.

™ The liquid is pumped to a heating zone, where it is

heated to supercritical conditions.
™ It then passes into the extraction vessel, where it
rapidly diffuses into the solid matrix & dissolves the
material to be extracted.

™ The dissolved material is swept from the extraction

cell into a separator at lower pressure, & the
extracted material settles out.

™ The CO2 can then be cooled, re-compressed &

recycled, or discharged to atmosphere.

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™ Analytes are trapped by letting the
solute-containing SF decompress
into an empty vial, through a
solvent, or onto a solid sorbent
material.

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 ™ Extractions are done in dynamic, static or combination
modes.

Dynamic Static Combination

The SF The SF circulates in a A static
continuously flows loop containing the extraction is
through the sample extraction vessel for performed for
in the extraction some period of time some period of
vessel & out the before being released time, followed
restrictor to the through the restrictor to by a dynamic
trapping vessel the trapping vessel extraction

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 Sample recovery
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Method to collect analytes after extraction

2 types :
1. Off-line collection
2. On-line collection
 Off-line collection

™ By immersing the restrictor in a few mL of solvent &

allowing the gaseous SF to escape into atmosphere
(solvent trapping).
OR
™ By trapping the analytes on adsorbent (i.e. silica) &
the adsorbed analytes are eluted with liquid solvent
(sorbent trapping).

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 On-line collection
™ The effluent from the restrictor, after depressurization,
is transferred directly to a chromatographic system i.e.
GC, SFC or HPLC.

™ Advantages :

9 Elimination of sample handling between the

extraction & the measurement.

9 Enhance sensitivity because no dilution of the

analytes occur.

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 Disadvantages
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™ Requires expensive equipment.

™ In practice, it is difficult to extract a wide range

of polarities.

™ Use of "modifiers" (e.g. methanol) with CO2

partially but not completely overcomes this
problem.
THE END

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