2. Supercritical fluid chromatography

3. Supercritical Fluid Chromatography
Contents
•
History
•
What is a supercritical fluid?
•
Theory of SFC
•
Instrumentation
•
Applications

4. History

The use of a supercritical fluid
mobile phase in chromatography
was first proposed in 1958 by J.
Lovelock.

The first actual report use of this in
a chromatographic system was in
1962 by Klesper et al, who used it
to separate thermally-labile
porphyrins.

5. S.F.C
•
S.F.C is a column chromatographic technique in which
supercritical fluid is used as a mobile phase.
•
Cost efficient
•
User friendly
•
Better resolution
•
Faster analysis

6. What is a supercritical fluid?
Supercritical Fluid exists 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.

7. Properties of Supercritical Fluid
•
High densities so they have a remarkable ability to dissolve
large, non-volatile molecules .
• Lower viscosities relative to liquid solvents.
• Inexpensive
• Ecofriendly
• Non-toxic.
• Higher diffusion coefficients and lower viscosities
relative to liquids.

8. 


SFC Advantages vs HPLC
Supercritical fluids have low viscosities
- faster analysis (5 to 10 X faster).
- the use of open tubular columns is feasible.
Column lengths from 10 to 20 m are used.
Resolving power is ~5X that of HPLC.

SFC Advantages vs GC
Can analyze non-volatile, polar, or adsorptive solutes without
derivatization.

Can analyze thermally labile compounds.

Can analyze solutes of much higher molecular weight.

9. Theory :




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 and 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 is similar to temperature programming in GC or
using a solvent gradient in HPLC.

10. SFC Instrumentation

Gas Supply or Mobile Phase

Stationary Phase

Pumps

Injectors

SFC Columns

Detector

Restrictor

12. Gas supply or Mobile Phase:
Liquid CO2 Pump
Cost
 interference with chromatographic
detectors
 physical properties like
nontoxic nature
nonflammable

are considered while selecting a
mobile phase.

13. Fluid
Critical Temperature (K) Critical Pressure (bar)
Carbon dioxide
304.1
73.8
Ethane
305.4
48.8
Ethylene
282.4
50.4
Propane
369.8
42.5
Propylene
364.9
46.0
Trifluoromethane (Fluoroform)
299.3
48.6
Chlorotrifluoromethane
302.0
38.7
Trichlorofluoromethane
471.2
44.1
Ammonia
405.5
113.5
Water
647.3
221.2
Cyclohexane
553.5
40.7
n-Pentane
469.7
33.7
Toluene
591.8
41.0

14. Carbon dioxide is the ideal to satisfy all the above properties.
 Safe to use
nontoxic
nonflammable
noncorrosive
inert.
 Detector compatible
• The main disadvantage of it is very polar or ionic compounds
are not able to be eluted.
• This can be overcome by adding a small portion of a second
fluid called a Modifier fluid. (alcohols, cyclic ethers, acetonitrile
and chloroform).


15. Modifiers



CO 2 is not a very good solvent for high molecular
weight, ionic and polar analytes
This can be overcome by adding a small portion of a second
fluid called modifier fluid
This is generally an organic solvent, which is completely
miscible with carbon dioxide
 methanol, acetonitrile, ethanol and 1-propanol.

16. Stationary Phase
Same as those for GC and LC, with some modification.
• Silica/Alumina
Useful for non-polar compounds
Lead to irreversible adsorption of some polar solutes
• Widely used polar Stationary Phase are
Polysiloxanes:- stable, flexible Si--O bond lead to
good diffusion.
Substituted with chemical groups for selective
interaction with analyte
Polymethylsiloxanes:- increase efficiency in
separating closely related polar analytes
Cyanopropyl polysiloxanes:-useful for compounds
with -COOH

17. Pumps:

Here mainly flow control is necessary.

So, syringe pumps are used for capillary
SFC for consistent pressure.

For Packed columns for easier blending
of the mobile phase or introduction of
modifier fluids reciprocating pumps are
used.
Syringe Pump

18. Injector
•


For packed column SFC, a conventional HPLC injection
system is adequate, but for the capillary column SFC, the
sample volume depends on column diameters and small
sample volumes must be quickly injected into the
column, therefore pneumatically driven valves are used.
Injector Volumes
Open tubular columns
◦ Injection volumes >96nL
◦ Greater volume affects resolution
Packed columns
◦ Injection volumes >1uL

19. Oven :
Conventional GC or LC ovens are used

20. SFC Columns
•
Open tubular (derived from GC)
-Large theoretical plates ~X500
-Easier to control pressure (low P drop)
•
Packed (derived from HPLC)
- Faster analysis
- Higher flow rates
- Higher sample capacity

21. Detectors
The choice of detectors will
depend upon
•
•
•
•
mobile phase composition
column type
flow rate
ability to withstand the high
pressures of SFC.

22. Contd..
•
•
•
It is compatible with both HPLC and GC detectors.
Flame photometric detectors
Flame ionization detectors
•
liquid-phase detectors like RID, ultraviolet-visible
spectrophotometric detectors and
•
light scattering detectors have been employed for SFC.

23. Back-Pressure Device or Restrictor
•
This is a device, which is used to maintain desired pressure in
the column by
- a pressure-adjustable diaphragm or
- controlled nozzle
so that the same column-outlet pressure is maintained
irrespective of the mobile phase pump flow rate.
• It keeps the mobile phase supercritical throughout the
separation and often must be heated to prevent clogging.
• The pressure restrictor is placed either after the detector or at
the end of the column.

24. Applications of SFC
By now SFC has been applied to wide variety of materials.








natural products
drugs
foods
pesticides
herbicides
surfactants
polymers and polymer additives
chiral compound

25. Natural products
•


separation of underivatized triterpene acids
estimation of caffeine from tea and conjugated bile acids
analysis of panaxadiol / panaxatriol in ginseng .
Pesticides
•
analysis of pesticide residues in canned foods, fruits and
vegetables wherein pyrethroids, herbicides, fungicides and
carbamates have been tested .

27. Surfactants
Separation of the oligomers in a sample of the nonionic
surfactant Triton X100 .
Lipids
for the analysis of high molecular weight lipids like
triacylglycerols.
Separation of fatty acid methyl esters, biosynthetic
polyunsaturated fatty acids (PUFA) 37 , nonsaponifiable
lipids , cholesterol and its esters in human serum and food
samples

28. Drugs






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Separation of drugs like
phenothiazine antipscychotics,
beta blockers
felodipine
clevidipine
methylated betacyclodextrins
vasodialators like isosorbide mononitrate, isosorbide
dinitrate, cyclandelate, nimodipine, amlodipine , oestrogens
combinations of various nonsteroidal antiinflammatory
drugs like flufenamic acid, mefenamic
acid, fenbufen, indomethacin mixtures, acetyl salicylic
acid, ketoprofen and fenbufen

29. Chiral compounds
SFC has been applied to separation of a large number of
enantiomers, diasterioisomers and geometrical isomers like
achiral and chiral analysis of camazepam and its
metabolites,
chiral separation of 1,3 dioxolane derivatives
Organometallics
Separation of metal chelates and organometals of thermally
labile category, chelates of transition metals, heavy
metals, organometallic compounds of lead, mercury and tin
has been carried out by SFC.

30. Conclusion
In the overall ranking of chromatographic techniques, it has
been judged that SFC falls somewhere between HPLC and
GC as the chromatographic method of choice. Lately SFC has
found a niche in the field of pharmaceutical chemistry and has
gained much support in the field of bioanalytical applications

31. References
http://www.chromatograhyonline.com/
 http://www.chemcenter/org
 http://www.kerouac.pharm.uky.edu/asrg/wave/wavehp.html
 http://www.anachem.umu.se/jumpstation.htm
 http://www.lplc.com/
 Instrumental analysis by Skoog Heller
 Instrumental methods of analysis by Kaur
 http://chrom.com/
 http://hplc.chem.vt.edu/
