Supercritical Fluid Chromatography

(SFC)
Introduction
SFC is a hybrid of gas and liquid chromatography
that combines some of the best features of both.

In SFC, the sample is carried through a separating column by
a supercritical fluid where the mixture is divided based on the
interaction between analytes and a stationary phase in the
column.

Mobile phase in SFC :
Fluid in supercritical state supercritical fluid
What is a supercritical fluid (SF) ?
SF is a material above its critical point (Pc and Tc).
It is not a gas, or a liquid, although it is sometimes referred to as a
dense gas.

SF generally exist at conditions above its critical temperature (Tc)
and pressure (Pc).



SF has densities, viscosities and other properties that are
intermediates between those of the substance in its gaseous and
liquid state
Tc: temperature above which a distinct liquid phase does not exist regardless of pressure
Pc: - minimum pressure required to liquefy a gas at its critical temperature
- vapor pressure at critical temperature
SF:

- diffusivity much higher than a liquid readily penetrates
porous and fibrous solids

- Low viscosity (equal to gas)
Phase diagram temperature/pressure of CO
2

Important Properties of Supercritical fluids (SF)

Remarkable ability to dissolve large non-volatile molecules
e.g., supercritical CO
2
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 CO
2
can be recovered by reducing the pressure and
allowing the CO
2
to evaporate

No need for organic solvents
environmentally friendly

Inexpensive, innocuous and non-toxic

Higher diffusion coefficients and lower viscosities (compared to liquid)
faster and higher resolution separations
Components of SFC
Fig. Schematic of a SFC installation using a HPLC packed column.
A commercial SFC installation
Novasep preparative SFC systems
- Cold liquid CO
2
is pumped
(1 2).

- Prior to entering the column, it is
heated and becomes supercritical
(23).

- Because of its low viscosity, the
pressure at the column outlet is
almost identical to the pressure at
the column inlet (3 4).

- At the column outlet, the mobile
phase is decompressed and
heated and becomes gaseous
(45)

- Products are recovered in
cyclones of appropriate design.

- The gaseous CO
2
is then
cleaned and cooled down and
returned to the tank.
SFC Columns
Two types of analytical columns used in SFC:

(1) Capillary columns of fused silica coated with cross-linked chemically bonded
stationary phases, that are used in GC are equally applicable in SFC.




(2) Packed columns developed for high performance liquid chromatography (HPLC)
are being used with SFC.
(a) Silica gel, the major material for current phases
Rigid porous (or nonporous) particles
Spherical particles
Irregularly-shaped particles


(2) Packed columns developed for high performance liquid chromatography (HPLC) are
being used with SFC.
(b) Bonded silica
Organochlorosilane
- If R is a polar functional group, the stationary phase will be polar.
i.e. cyano (C
2
H
4
CN), diol (C
3
H
6
OCH
2
CHOHCH
2
OH), or amino
(C
3
H
6
NH
2
)
- If R is a non polar functional group, the stationary phase will be non polar.
i.e. n-octyl (C8) or n-octyldecyl (C18) hydrocarbon chain

(c) Other stationary phases of varying polarity
Aluminium oxide Al
2
O
3

Zirconium oxide ZrO
2

Styrene-divinylbenzene
Hydroxymethylstyrene
Porous graphite
SFC Mobile Phase
CO
2
is the primary mobile phase used in SFC.

The advantage ofCO
2
as the mobile phase is
- low cost,
- low interference with chromatographic detectors,
- nontoxic,
- low critical temperature (31.1
o
C),
- inflammability and that it can permit a flame ionisation detector to be used, with all
the benefits in terms of ease of use, linearity and sensitivity

Disadvantage of carbon dioxide is
- inability to elute polar or ionic compounds.
By adding a small portion of a second fluid, modifier, this can be overcome. Modifiers
are generally an organic fluid (such as alcohols ,cyclic , etc) which are completely
miscible with carbon dioxide. Modifiers improve the solvating ability of the SCF and
sometimes enhance separation selectivity
Typical Supercritical mobile phase
SFC Injection

-For packed SFC, a typical LC injection valve is commonly used
load
Inject
- In capillary SFC, small sample volumes must be quickly injected into the column and
therefore pneumatically driven valves are used.
Carrier
Waste/Drain
Sample
in
Column
Six-way injection valve
Injection of identical volume of sample
Loading Injection
SFC Pump
The type of high pressure pump used in SFC is determined by the column type.

- For packed columns, reciprocating pumps are generally used








Reciprocating pumps allow easier mixing of the mobile phase or introduction of modifier
fluids.

- For capillary columns, syringe pumps are used.
Syringe pumps provide consistent
pressure for a neat mobile phase.
Double-plunger micro pump (Reciprocating pump)
Stroke length 1.0 mm
Plunger diameter 2.5 mm
Stroke volume 5 ml/stroke

Pumping mechanism
dual head
reciprocation
SFC Detector

FID (flame ionization detector) and MS detector.
The restrictor is installed before the detector,

UV-absorption, fluorescence or light diffusion.
The restrictor is installed after the detector.
Restrictor: a device installed in the SFC system to control pressure of
the fluid during the chromatographic process
The major difference in SFC and conventional LC equipment is
the pumping systems as well as the safety features installed to maintain higher
pressure.

Unique SFC equipment differences are:

1. Carbon dioxide tank for mobile phase supply
- Equipped with a pressure relief value and rupture disk

2. High-pressure pump
- Chiller to maintain mobile phase in liquid state

3. High-speed injector

4. Pressure restrictor
- High-pressure tubing

5. High-pressure flow cell for UV detection

6. Solvent collection device with ability to vent to a laboratory hood or elephant trunk.
SFC Vs LC Instrumentations
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
< up to 25% of all separation problems fall into this category
< 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
Faster elution
Reduction in
peak width
Reduction in
elution time
Reduction in
peak width
Reduction in
elution time
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
SFC is more and more used in research and development laboratories
and pilot plants of the pharmaceutical and fine chemical industries.

SFC is particulary interesting for the purification of :
chiral compounds actives or intermediates from complex mixtures lipophilic
compounds

Application of SFC
The ability to vary selectivity by programming the parameters P
(pressure) and T (temperature) rather than by modifying the chemical
composition of the eluent.

The range of compounds analysed by SFC includes lipids and oils,
emulsifiers, oligomers and polymers (compounds of molecular mass
greater than 1000 which cannot be studied in GC)
(1) Chiral separation
The use of supercritical fluids to separate enantiomers is one of the most
important tasks in several areas of research, especially pharmaceuticals and
agrochemicals.

It is well known that the two enantiomeric forms of a molecule can display
dramatically different biological activity.
Example : Chiral separation of four triazole
pesticides by supercritical fluid chromatography
- Showed the effects of
different organic modifiers on
the resolution
and retention via k value
k : 1 - 5
R greater than 1.5

Good if
(2) Polymer separation
Column temperature: 80
0
C,
Pressure: 19.6 MPa.
Coulmn: Inertsil Ph-3
Consistent flow rate of CO
2
: 3.0 ml/min
Polyprenol or 1,4-polyprenyl alcohols has 30 monomers
(3) High throughput screening of pharmaceuticals