1. SSuuppeerr CCrriittiiccaall FFlluuiidd
CChhrroommaattooggrraapphhyy
Presented by,
B.BHAVYA,
No. 256213886005,
M.Pharm (Pharmaceutics).
Under The Guidence Of
Mr. Uttam Prasad
M.pharmacy.

2. HISTORY
• Use of SUPER CRITICAL FLUID
CHROMATOGRAPHY (SFC) was first proposed
in 1958 by J.LOVELOCK
• First Reported use was in 1962 by KLESPER
et.al., who separated thermally liable porphyrins.

3. DEFINITION
• A supercritical fluid
chromatography is a material
that can be either liquid or
gas used in state above
critical temperature or critical
pressure where gases and
liquid can coexist.
• Supercritical fluid have a
viscosity close to gases a
density close liquids and a
high diffusivity.

4. What is critical?
• Critical temperature is temperature above which a
distinct liquid phase cannot exist regardless of pressure.
• The vapor pressure of a substance at its critical
temperature is its critical pressure.
• Carbon dioxide is known to be most stable and an
excellent solvent compound and is normally used in
mobile phase for super critical fluid chromatography.
• High densities so have a remarkable ability to dissolve
large non-volatile molecules.
• Lower viscosities relative to liquid solvents.
• Inexpensive, ecofriendly and nontoxic.

6. Contents
• Introduction
• Instrumentation:
Injectors
Columns
Stationary and Mobile PHASE
Detectors
• Advantages
• Disadvantages
• Comparison
• Application
• Conclusion

7. Introduction
• In SFC Sample is carried through a separating column by an
SFC fluid where mixture is divided into unique bands based on
amount of interaction between industrial analytes and stationary
phase in the column. As these bands leave columns there
identities and quantities are determined by detector.
• SFC TEMPERATURE : For every substance there is a
temperature above which it can no longer exist as a liquid no
matter how much pressure is applied.
• SFC PRESSURE : For every substance there is a pressure
above which it can no longer exist as a liquid no matter how high
temperature is raised.

8. Instrumentation
• GAS SUPPLY OR MOBILE PHASE
• STATIONARY PHASE
• PUMPS
• INJECTORS
• OVEN
• SFC COLUMN
• DETECTOR
• RESTRICTOR

10. Pumps
• Flow controlling is the vital function of pumping systems
• In contrast to HPLC pumping systems pressure rather than flow
control is necessary and pulseless operation is more important.
• In general type of high pressure pump used in super critical fluid
is determined by column type.
• For packed columns for easier blending of mobile phase or
introduction of modifier fluids reciprocating pumps are generally
used.
• For capillary super critical fluid syringe pumps are most
commonly employed.
• Reciprocating pumps allow easier mixing of mobile phase or
introduction of modifier fluids.
• Syringe pumps provide consistent pressure for neat mobile
phase.

11. Oven
• A thermostated column oven is required for precise
temparature control of the mobile phase. Conventional GC or
LC ovens are generally used.
Back – Pressure Device or Restrictor
• This is a device which is used to maintain desired pressure in
column by pressure adjustable diaphragm or controlled nozzle, so
that, same column outlet pressure is maintained irrespective of
mobile phase pump flow rate.
• It keeps mobile phase super critical throughout separation and
often must be heated to prevent clogging
• Pressure restriction is placed either at the end of the column or
after detector.

12. Types of injectors
INJECTOR : For Packed SFC a conventional HPLC injection system is
adequate, but for a capillary column SFC, the small volume depends on
column diameters and small volume must be quickly injected into
column.
Therefore pneumatically driven values are used.
LOOP INJECTORS : It is a direct transposition of what is applied in
analytical SFC
Used mostly for preliminary tests of column performance and elution
parameters.
INLINE INJECTION : More versatile
• System offers better flexibility for changing injected volume.
• High pressure pump required to inject feed solution.
• Injected stream dissolves in diluent flow.
INCOLOUMN INJECTION : An alternative system
• Permits injection of feed solution directly onto column
• No dilutions required

13. Loop injector
In line injector
In column injector

14. Columns
• Once the sample is injected into supercritical stream, it is carried into
the analytical column that contains a highly viscous liquid (stationary
phase) into which the analytes can be temporarily adsorbed and then
released based on their chemical nature. This temporary retention
causes some analytes to remain longer in the column and is what
allows the separation of the mixture different types of stationary phases
are available with varying compositions and polarities
 There are two types of analytical columns used in SFC ,
A) Packed columns contain small deactivated particles so which the
stationary phases adheres the columns are conventionally stainless
steel
B) Capillary columns are open tubular columns of narrow internal
diameter made of fused silics with the stationary phase bonded to the
wall of the column

16. Stationary phases
• Same as those used for GC & LC with some modifications.
Silica / Alumina
• Useful for nonpolar compounds.
• Lead to irreversible adsorption of some polar solutes.
• Bonded to provide less adsorptive packing material.
• Need organic modifiers to elute analytes.
Widely used polar stationary phase:
• POLYSILOXANE – Stable, Flexible, Si-O bond leads to
good diffusion.
• POLYMETHYL SILOXANES – Increase efficiency in
separating closed related analytes.

17. Mobile phase or Gas supply
• Cost, interference with chromatographic
detectors and physical properties like nontoxic
nature,nonflamable,low critical values are
considered while selecting mobile phase.
• Carbondioxide : is the ideal to satisfy all the
above properties.
• Safe to use nontoxic , nonflamable , inert ,
Noncorrosive.
The main disadvantage is it is very polar
or ionic compounds are not able to be eluted.

18. • This can be overcome by adding a small portion of
a second fluid called a modifier fluid. This is
generally an organic fluid which is completely
miscible with carbon dioxide (alchols,cyclic ethers)
but can be almost any liquid including water.
• The addition improves solvating ability of
supercriticalfluid and sometimes enhances
selectivity of seperation efficiency by blocking
some of the highly active sites on the stationary
phase.modifier fluids are commonly
used,especially in packed column sfc.

19. Other SFC solvents
• Nitrous oxide : Similar in solvating and
separation properties to CO2.
But its expensive.
• Alkanes : Less safe and not as detector
compatible than CO2
- better solvent characteristics for nonpolar
solutes.
• Halocarbons , xenon etc. : Specialty applications
only.
More polar solvents for highly polar &high
molecular weight compounds.

20. Detectors
• Optical, flame & spectroscopic detectors
• Choice depends upon:
Mobile phase composition
Column type
Flow rate
Ability to withstand high pressure of SFC.
It is compatible with both HPLC and GC detectors.
Flame ionization Process
Liquid phase detectors like RID, Ultra violet – Visible
Spectrophotometric detector and Light Scattering
detectors have been employed for SFC.

22. Advantages
• Lower operating temperatures.
• Improved yield.
• Improved product properties.
• Favorable combination of process steps.
• Easier regeneration of supercritical solvent.
• Lower product cost.
• Very high volatility compared to dissolved substance.
• Provide rapid separation without use of organic solvents.
• Uses environmentally conscious technology.
• High resolution at lower temperature.
• High diffusion coefficient.
• Low Viscocity.

23. Disadvantages
• Elevated pressures required.
• Relative high cost of equipment.
• Unusual operating conditions.
• Complicated phase behaviour.
• Expensive technology.
• Cleaning will be time consuming.

24. SFC Vs HPLC
 SFC has low viscosities.
 Faster analysis ( 5*10X* Faster).
 Use of open tubular columns in feasible column lenghth
from 10 – 20m is possible.
 Resolving power is ~5X that of HPLC.
SFC Vs G.C
 Can analyze non volatile polar or adsorptive solvents
without derivatization.
 Can analyze solutes of much higher molecular weights.

25. Mix Fluid
 Addition of polar organic modifier.
Advantages:
• Enhance solubility of polar analyte.
• Reduce retention volume.
• Eliminate strong interaction between adsorptive site and polar site.
• Will modify polarity of diluent.
• CO2 – Ideal to satisfy all properties.
• Safe to use.
• Nontoxic.
• Non flammable.
• Non corrosive.
• Detector compatible.
Disadvantages:
• Very polar or ionic not able to be eluted.
• Can overcome by adding small portion of scanned fluid called
modifier fluid (Alcohols, cyclic ethers, acetonitrile, chloroform).

26. Applications
 SFC finds use in industry primarily for separation of chiral molecules
and uses the same columns as a standard HPLC systems
 SFC is now commonly used for achiral separations and purifications in
the pharmaceutical industry
 Fossil fuel and hydrocarbons.
 Polymers.
 Drugs and pharmaceuticals.
 Explosives and propellants.
 Lipids.
 Carbohydrates.
 Industrial chemicals.
 Foods and flavours.
 Natural products.
 Metal chelates and organoleptic compounds.
 Enantiomers.

27. Applications
NATURAL PRODUCTS :
• To determine the origin of oil and improved possibilities of
determination of relations between oil constituents and physical as well
as biochemical properties of oil.
• Separation of salt and common free bile acids like ursodeoxycholic acid
and chenodeoxycholic acid in pharmaceutical preparation has been
reported using phenyl bonded silica column and SFC –CO2 modified
with methanol.
• Capillary SFC has been used for analysis of panaxadiol paraxatriol in
ginseng and its preparation, vegetable carotenoids and pyrolizidine
alkaloids.
PESTICIDES :
• SCF Extraction & Chromatography has been used for analysis of
pesticide residues in canned foods, fruits, vegetables where in
pyrethroids, herbicides, fungisides and carbamates have been tested

28. Surfactants:
• Separation of the oligomers in sample of nonionic surfactant triton X 100 has
been reported where detected by measuring the total ion current produced
by the chemical ionization mass spectrometer.
Lipids :
• SFC has also been applied to analyze phospholipids after conversion to
diacylglycerol derivatives, fatty acids, methyl esters, biosynthetic poly
unsaturated fatty acids (PUFA), non saponifiable lipids, cholesterol and its
esters in human serum, mono, di and tri glycerides in pharmaceutical
excipients has been carried out by SFC successfully.
Drugs :
• Modern drug substances are commonly nonvolatile and thermally or
chemically liable therefore analysis by HPLC is common over G.C
• In SFC conditions are mild and no volatilization is required, therefore there is
a possibility for better results through SFC than any existing method.
• Seperation of various categories of drugs like antidepressants
phenothiazines of various categories of drugs like antidepressants,
phenothiazines, antipsychotic, beta blockers etc

29. • In overall ranking of chromatographic
techniques it can be judges that SFC falls
somewhere between HPLC and GC.
• In field of pharmaceutical chemistry and
bioanalytical applications SFC gained its
applications.