Super Critical Fluid Chromatography

Super Critical Fluid
Chromatography
Mr. Sanket P. Shinde
Assistant Professor
Pune-Maharashtra.

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Introduction
o Supercritical fluid extraction and Supercritical fluid chromatography are
techniques which use supercritical fluids as solvent for both extraction
and separation respectively.
o The properties such as density, viscosity and diffusion constant of the
supercritical fluids are intermediate between those of a substance in
gaseous and liquid state.
o This helps in efficient extraction and chromatographic separation
compared to other techniques.

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Principle
The basic principle of SFE and SFC is almost same. Both techniques use
supercritical fluids as the solvent for extraction and separation.
Super Critical Fluids
Supercritical fluid can be described as a fluid that obtained by heating above
the critical temperature and compressing above the critical pressure.
Critical Temperature
Above certain temperature the substance can no longer exist as a liquid,
irrespective of the increase in pressure. That temperature is called as critical
temperature.
Critical Pressure
Above certain pressure the substance can no longer exist as a gas irrespective
of the increase in temperature. That point is called as critical pressure.

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o A substance can exists as solid or liquid or gas under various temperature
and pressure.
o When the temperature is increased, solids become liquid above their
melting point.
o There may be direct conversion of solid to vapor state by the process
sublimation.
o Similarly liquids become gases above their boiling point.
o However above the critical temperature and critical pressure the substance
neither acts as liquid or gas irrespective of increase in temperature or
pressure. This point is called as critical point.
o At critical point, the density of the liquid and gas is same and the fluid
cannot be liquefied by increasing the pressure.
o Above the critical point the state of the substance is said to be supercritical
fluid where no phase change occurs.
Continue…

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Phase Diagram

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o SCF is produced from liquid by increasing temperature at constant
pressure or from gas by increasing pressure at constant temperature.
o Supercritical fluid can also be termed as compressed liquid or a
compressed gas.
o Separation in SFC is based on the density of the supercritical fluid
which corresponds to solvating power.
o As the pressure in the system is increased, the density of the
supercritical fluid increases and correspondingly its solvating power
increases.
o This in turn shortens the elution time for the eluent as pressure changes
in SFC have a pronounced effect on the retention of analytes.
o This effect is general and similar to programmed temperature in GC or
gradient elution in HPLC.
Continue…

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Properties of Super Critical Fluids (SCFs)
1. Supercritical fluids have combined properties of gases and liquid
thermal conductivity and diffusivity of a SCF are higher than the liquid
whereas its viscosity is much lower even though density is same
supercritical fluids generally have increased solubility.
2. SCFs have high densities (0.2-0.5gm/cm3). When density of the SCFs is
increased solubility also increases thus SCFs can able to dissolve large,
non-volatile molecules.
3. Recovery of dissolved analytes can be easily done by allowing the
solutions to equilibrate with the atmosphere at low temperatures.
4. The SCFs are miscible with permanent gases such as Nitrogen or
Hydrogen thus the concentrations of dissolved gases in the fluid are
high.

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o Supercritical fluid extraction (SFE) is a technique which is used to separate
one component or extractant from a mixture or matrix using supercritical
fluids as the extracting solvent.
o Solid samples and even liquid samples also can be used for extraction.
o Supercritical Fluid Extraction (SFE) is based on the fact that, near the
critical point of the solvent, its properties change rapidly with only slight
variations of pressure.
o Generally Carbon dioxide is widely used as supercritical fluid for extraction.
o The supercritical properties of CO2 above its critical temperature of 31°C
and critical pressure of 74 bars are utilized for SFE.
o Unlike other extraction technique SFE does not leave residual solvent in the
final extract.
Supercritical Fluid Extraction (SFE)
Introduction

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Instrumentation
Schematic diagram of supercritical fluid extraction

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The extraction in SFE is done by two modes
1. Static extraction
In the static extraction experiment, there are two distinct steps in the process
a. The mobile phase fills the extraction cell and interacts with the sample.
b. Second pump is opened and the extracted substances are taken out at once.
o In order to choose the mobile phase for SFE, parameters taken into
consideration include the polarity and solubility of the samples in the
mobile phase.
o Carbon dioxide is the most common mobile phase for SFE.
o It has a capability to dissolve non-polar materials like alkanes.
o For semi-polar compounds CO2 can be used as a single component mobile
phase.
o However, for compounds which have polar characteristic, supercritical
carbon dioxide must be modified by addition of polar solvents like
methanol.
o These extra solvents can be introduced into the system through a separate
injection pump

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2. Dynamic extraction
o In dynamic extraction, the second pump sending the materials out to the
collection chamber is always open during the extraction process.
o Thus, the mobile phase reaches the extraction cell and extracts components
in order to take them out consistently.
Extraction vessels
o These are the vessels in which samples for extraction is placed.
o These vessels may be of simple tubing or sophisticated vessels with quick
release fittings.
o The minimum pressure required for extraction is at least 74 bars.
o These vessels should withstand the high pressure applied during extraction
of samples such as vegetable oils for which a pressure of around 800 bars is
applied.

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Extraction modes
There are two modes in terms of collecting and detecting the components
1. Off-line extraction
o Off-line extraction is done by taking the mobile phase out with the
extracted components and directing them towards the collection chamber.
o At this point, supercritical fluid phase is evaporated and released to
atmosphere and the components are captured in a solution or a convenient
adsorption surface.
o Then the extracted fragments are processed and prepared for a separation
method.
o This extra manipulation step between extractor and chromatography
instrument can cause errors.

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2. On-line extraction
o The on-line method is more sensitive because it directly transfers all
extracted materials to a separation unit, mostly a chromatography
instrument, without taking them out of the mobile phase.
o In this extraction/detection type, there is no extra sample preparation after
extraction for separation process.
o This minimizes the errors coming from manipulation steps.
o Additionally, sample loss does not occur and sensitivity increases.

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Advantages of SFE
1. The extraction process provides solvent free extracts thus devoid of
residual solvent issue of other extraction techniques.
2. Supercritical fluids are inexpensive thus the technique is cost effective.
3. CO2 is non-toxic, nonflammable, odorless, tasteless, inert thus safe for
use.
4. Provides rapid extraction without use of much organic solvents thus
environmentally friendly.
5. Disposal costs are much less and in industrial processes, the fluids can be
simple to recycle.

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1. It is useful for the extraction of natural products such as plants and algae.
Example: extraction of ginger.
2. It is useful in extraction of natural oils from crude powders.
3. SFE can be used as a sample preparation step for analytical purposes.
4. It is used to remove unwanted material from a product (e.g. decaffeination)
or collect a desired product (e.g. essential oils).
5. It finds its use in food, aromas, essential oils and nutraceutical industries.
6. SCFs are finding applications in fractionation of low vapour pressure oils, in
several reactions in different areas of biochemistry, polymer chemistry,
environmental sciences, polymer and material industries.
Applications of SFE

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o SFC is a separation technique that uses supercritical fluids as mobile phase
for the separation of one or more components from a mixture on a
stationary phase.
o It is a hybrid of gas and liquid chromatography.
o The mobile phase acts as a liquid when it is below its critical temperature
and above its critical pressure.
o It acts as gas when the mobile phase is above its critical temperature and
below its critical pressure.
o Thus the supercritical state is neither gas nor liquid.
o The stationary phase is usually the SFC columns. This technique is more
versatile than high performance liquid chromatography.
o It is a rapid, user friendly and cost efficient technique with high throughput
and precision compared to other liquid chromatographic methods. It gives
better resolution than HPLC.
o The instrumentation of SFC is versatile with multi-detectors compatibility.
Due to these advantages SFC becomes more useful technique in industries.
Supercritical Fluid Chromatography (SFC)
Introduction

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The instrumentation of SFE and SFC is almost similar except the extraction
vessel in case of SFE is replaced with analytical column in SFC.
The instrument is similar to HPLC and the components are given below.
1. Mobile phase reservoir
2. Cooler
3. Pump
4. Injector
5. Thermo stated oven
6. Columns
7. Restrictor Back pressure regulator
8. Microprocessor
9. Detector
10. Collector
11. Data reader
Instrumentation

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Flow diagram of construction of supercritical fluid chromatography

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1. Mobile phase reservoir
o This is usually a stainless steel cylinder when gases are used.
o For example, the widely used CO2 is usually stored in the cylinder.
o The cylinder is provided with pressure regulator to control the flow of gas.
2. Cooler
o Cooler is used for to cool the CO2 before pumped into the extraction
vessel or column.
o Usually CO2 is pumped below 5°C and at a pressure of 50 bars.
o The solvent is pumped as liquid in cooling condition. For this purpose
cooler is used.

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3. Pumps
o Generally for small scale equipments reciprocating pumps or syringe pumps
are used whereas diaphragm pumps are used for large scale.
o In case of SFC, pumps are chosen depending upon the types of column.
o For packed columns, reciprocating pumps and for capillary SFC, syringe
pumps are most commonly used.
o With reciprocating pumps the mixing of mobile phase is easier especially
when modifiers are introduced, whereas syringe pumps provide consistent
pressure for a neat mobile phase.
o In contract to HPLC pumping system, pressure rather than flow control is
necessary and pulse less operation is more critical.

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4. Injector
o Injection in SFC is usually achieved by switching of the content of a sample
loop into the carrier fluid at the column entrance by means of a suitable
valve.
o For packed column SFC, a conventional HPLC injection system is adequate,
but for the capillary column SFC, pneumatically driven valves are used as
the small sample volumes must be quickly injected into the column quickly.
5. Thermostated Oven
o A thermostated column oven is employed for maintenance of precise
temperature of the mobile phase.
o The column in case of SFC and extraction vessel in case of SFE is placed
inside the oven.
o The temperature display with keypads is provided for adjustment of
temperature.
o The cooled fluid from pump should be heated to achieve supercritical state
before entering into the column or extraction vessel.

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6. Columns
o Columns are similar to HPLC, There are two categories of columns are used.
o One is packed SFC columns and the other is capillary columns.
o The stationary phases used in column should not be soluble with supercritical
CO2.
o Stationary phases such as octadecylsilyl (C 18) or aminopropyl bonded silica,
Decylsilyl C8 are used with SFC.
a) Mobile Phase
o The mobile phase can be selected from the solvent groups of inorganic
solvents, hydrocarbons, alcohols, ethers, halides; or can be acetone,
acetonitrile, pyridine, etc.
o However, the most common supercritical fluid which is used in SFC is
carbon dioxide because it is;
• Readily available and Inexpensive
• It has an accessible critical point and Relatively safe
• Considered green since it has been recycled
• Miscible with a wide range of highly polar modifiers.

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b) Modifiers
o Today, most SFC applications are performed on relatively polar stationary
phases with CO2 modified with an organic solvent and sometimes other
highly polar components, such as acids and bases, called additives or
modifiers.
o Modifiers can also enhance selectivity of separation and improve separation
efficiency by blocking some of the highly active sites on the stationary
phase.
o Methanol is by far the most widely used modifier and among the most polar
modifiers completely miscible with CO2.
o Advantages of methanol include:
• Availability,
• Inexpensiveness,
• Complete miscibility with CO2,
• Low UV cut-off (about 205 nm) and relatively low toxicity.

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7. Restrictor or Back-Pressure Device
o This device is used to maintain the pressure the mobile phase above the
critical point.
o It consists of a pressure-adjustable diaphragm or controlled nozzle which
adjust the pressure in the column or the extraction vessel.
o With the help of this device the column-outlet pressure is maintained
irrespective of the mobile phase pump flow rate.
o The restrictor not only determines the back pressure but also controls the
mass flow rate of the supercritical fluid flowing through the system.
o The pressure restrictor is placed either after the detector or at the end of the
column.
o There are two types of restrictors are in use.
a) Simple capillary restrictor
b) Mechanical restrictor

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a) Simple Capillary Restrictor
o It is used for capillary SFC. It consists of a narrow bore fused silica
capillary tubing.
o The pressure regulation is achieved through the flow resistance brought
about by the length and diameter of the tube.
o The greater pressure maintenance is achieved with more length and narrow
tube.
o This restrictor are used SFC coupled with gas detectors of SFE with small
volume extraction vessels.
b) Mechanical Restrictor
o It is suitable for the high mass flow rate of packed column system with UV
detector.
o It consists of mechanically adjustable valve or pneumatic system which can
be electronically controlled.

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8. Microprocessor
o There may be one or more Microprocessors are used to control the
variables such as pumping pressures, oven temperature and detector
performance.
9. Detector
o The choice of detectors for SFC is depending upon the mobile phase
composition, column type, flow rate and ability to withstand the high
pressures. Detectors of SFC are similar to HPLC or GC.
o The detectors which are generally used with GC such as flame ionization
detectors and flame photometric detectors can be used conveniently.
o Similarly detectors which are used in liquid chromatography such as,
ultraviolet-visible spectrophotometric detectors refractive index detectors
and light scattering detectors have also been employed.
o SFC is also compatible with mass spectrometry, FTIR detectors,
fluorescence emission spectrometer and thermionic detectors.

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10. Collector
o Collecting devices are usually associated with SFE.
o The extract is obtained as fine particles through a aerosol like device and
collected.
o In SFC the mobile phase or modifier which reaches the detector may go to
waste through the detector outlet.
11. Data reader
o Read out devices is usually a computing system with suitable software
that can display the chromatogram and other parameters.
o The calibration and system suitability parameters can conveniently
determined using the software's.

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o In SFC, CO2 is cooled by the help of cooler and introduced into the column
which is placed in the oven.
o The temperature is maintained by the temperature programming to achieve
the critical point.
o The restrictor maintains the pressure thus the mobile phase is converted to
supercritical fluids.
o The sample is carried through a separating column by supercritical fluid
where the mixture is divided into unique bands based on the amount of
interaction between the individual analytes and the stationary phase in the
column.
o The separated analytes reach the detector, the signals are detected and their
identities and quantities are determined.
Working

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Advantages of SFC
1. The less viscosity and higher diffusivity of SCFs provides the ability to
increase column lengths, faster flow rates, higher theoretical plates thus
efficient separation.
2. With the advanced of SFC-MS, even picogram per milliliter
concentrations can be detected easily which is not possible with other
techniques.
3. SFC-MS shows great potential in the field of bioanalytical chemistry, but
especially in chiral separation and detection.
4. SFC has got multi detector compatibility which allows the analysis of
thermolabile compounds.
5. SCFs are inexpensive, safe and non-toxic higher diffusion constants and
lower viscosities relative to liquid solvents.
6. Due to higher diffusion coefficient means rapid analysis is possible
compare to HPLC.

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1. The separation, identification and quantification of phytoconstituents from
crude extracts can be done by SFC.
2. It is used for the identification and quantitation of pesticides in food
products like canned foods, vegetables and fruits.
3. It is used for the analysis of phospholipids and high molecular weight
lipids like triacylglycerols.
4. It is useful in the determination of polymers and high molecular weight
compounds.
5. It is useful for the separation and estimation of drug molecules in bulk and
pharmaceutical formulations.
6. It is useful in bioanalytical techniques for the analysis of drug level in
blood plasma, serum and urine.
7. It has been applied for the separation of a large number of enantiomers,
diastereoisomers and geometrical isomers.
8. SFC finds its use in impurity profiling while it is coupled with mass
detector.
Applications of SFC

Super Critical Fluid Chromatography

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