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1. SUPERCRITICAL FLUID
CHROMETOGRAPHY AND FLASH
CHROMATOGRAPHY :- PRINCIPLE
AND APPLICATION
PREPARED BY:- ANKIT S. SHAH
(09PGPH010)

2. SUPERCRITICAL FLUID CHROMATOGRAPHY
 Chromatography:- separation technique of complex
chemical mixtures into individual components .
 Various techniques.
 Chromatography – mobile phase
Gas chromatography - Gas
Liquid chromatography - Liquid
Supercritical fluid - Supercritical fluid
chromatography

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

4. What is SUPERCRITICAL FLUID?
 SCF can be described as a fluid obtained by heating above the critical
temperature and compressing above the critical pressure .
Phase Diagram for Pure Substance

5.  For every substance, there is a temperature above which it can
no longer exist as a liquid, no matter how much pressure is
applied.
 Likewise, there is a pressure above which the substance can
no longer exist as a gas no matter how high the temperature is
raised.
 These points are called critical temperature and critical
pressure respectively.
 Above this point,the substance acts as a supercritical fluid .

6. PROPERTIES AND ADVANTAGES OF SCF
 high densities so they have a remarkable ability to dissolve
large, non-volatile molecules .
 dissolved analytes can be easily recovered by simply allowing
the solutions to equilibrate with the atmosphere at low
temperatures . So useful with thermally unstable analytes.
 inexpensive
 Ecofriendly
 non-toxic

7. PROPERTIES AND ADVANTAGES OF SCF
 Lower viscosities relative to liquid solvents.
 Greater diffusibility means longer column length can be used.
 Higher diffusion coefficient means higher analysis speed that
Comparison of properties of gas,SCF and liquid
Gas (STP) SCF Liquid
Property
Density (g/cm 3 ) (0.6-2) x 10 -3 0.2-0.5 0.6-2
Diffusion coefficient (1-4) x 10 -1 10 -3 x 10 - 4 (0.2-2) x 10 -5
(cm 2 /s)
Viscosity (G Cm -1 s -1 ) (1-4) x 10 - 4 (1-3) x 10 - 4 (0.2-3) x 10 -2

8.  The two supercritical fluids of particular interest are , carbon
dioxide and water.
 Carbon dioxide :-
 non-flammable,
 nontoxic
 low critical temperature of 31.9 C and moderate
critical pressure of 73bar.
 It is miscible with variety of organic solvents and is
readily recovered after processing.
 It diffuses faster than conventional liquid solvents.

9.  Water .
 critical temperature of 647K and critical pressure of 220bar
due to its high polarity.
 The character of water at supercritical conditions changes
from one that supports only ionic species at ambient
conditions to one that dissolves paraffins, aromatics, gases and
salts.
 Due to this unique property, research has been carried out on
supercritical water for reaction and separation processes to
treat toxic wastewater.

10.  The final choice of SCF depends on the specific
application as well as other factors like safety,
flammability, phase behavior, solubility at operating
conditions and cost of fluid.

11. PRINCIPLE

12. INSTRUMENTATION
 The instrumentation of SFC is similar in most regards to
instrumentation for HPLC because the pressure and
temperature required for creating supercritical fluid from
several gases or liquids lie well within the operating limits of
HPLC equipment
 However, there are two main differences between the two.
 a thermostated oven required to provide precise temperature
control of the mobile phase
 a restrictor to maintain the pressure in the column at a
desired level and to convert the eluent from SCF to a gas for
transfer to detector .

13. Flow Diagram of Construction of SFC Instrument

14.  the mobile phase is pumped as a liquid and is brought into the
supercritical region by heating it above its supercritical
temperature before it enters the analytical column.
 It passes through an injection valve where the sample is
introduced into the supercritical stream
 It is maintained supercritical as it passes through the column
into the detector by a pressure restrictor.

15.  Mobile phase:-
 There are a number of possible fluids, which may be used in
SFC as a mobile phase.
 However, based on its low cost, low interference with
chromatographic detectors and good physical properties
(nontoxic, nonflammable, low critical values) CO2 is the most
used mobile phase for SFC .
 excellent solvent for a variety of nonpolar organic molecules.

16.  Columns
 Basically two types of analytical columns are used in SFC,
packed and capillary.
 Earlier work employed absorbents such as alumna, silica or
polystyrene .
 More recent packed column work has involved bonded
stationary phases such as octadecylsilyl (C 18 ).
 Oven
 A thermostated column oven is required for precise
temperature control of the mobile phase.

17.  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.

18.  Microprocessor
 The commercial instruments for SFC are ordinarily equipped
with one or more microprocessors to control such variables as
pumping pressures, oven temperature and detector
performance.
 Detector
 it is compatible with both HPLC and GC detectors.
 flame photometric detectors
 flame ionization detectors

19.  refractive index detectors
 ultraviolet-visible spectrophotometric detectors
 light scattering detectors
 The choice of detectors will depend upon the mobile phase
composition, column type, flow rate and ability to withstand
the high pressures of SFC.

20.  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.

21. COMPARISION WITH OTHER TYPES OF
CHROMATOGRAPHY:-
 Several physical properties of SCF are intermediate between
gases and liquids.
 SFC is inherently faster than LC because the lower viscosity
makes use of higher flow rates.
 ability to separate thermally labile compounds (20% drugs)
 SFC is faster than HPLC, because of its lower viscosity and
higher diffusion rates
Unlike GC, by changing the mobile phase the selectivity can
be varied in SFC .

22.  Due to the thermally unstable or non- –volatile nature of many
nitrogen and / or sulfur containing compounds, they cannot be
analyzed by GC . Because SFC generally uses carbon dioxide,
collected as a byproduct of other chemical reactions or is
collected directly from the atmosphere, it contributes no new
chemicals to the environment.
 biggest advantage that SFC has over HPLC lies within the
differences in the mobile phases.
 Supercritical fluids are less viscous, possess a higher
diffusivity than liquids under HPLC conditions.This provides
not only the ability to increase column lengths, but also allows
for faster flow rates.

23.  SFC can be set up for sub ambient temperatures, which has
been key in many chiral separations .

24. APPLICATION 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. n Natural Products
 Lipophilic – amphiphilic compounds with properties
between volatiles and hydrophilic compounds often create
problems in connection with their isolation and analytical
determination resulting in an analytical gray area,
 But SFC has been found to give relatively fast and simple
procedures for determination of oil constituents such as
chlorophyll and its derivatives .
 Separation of bile salts and common free bile acids like
ursodeoxycholic acid and chenodeoxycholic acid in
pharmaceutical preparation.

26.  separation of underivatized triterpene acids
 estimation of caffeine from tea and conjugated bile acids
 analysis of panaxadiol / panaxatriol in ginseng .
2) Pesticides
 analysis of pesticide residues in canned foods, fruits and
vegetables wherein pyrethroids, herbicides, fungicides and
carbamates have been tested .

27. n Surfactants
 Separation of the oligomers in a sample of the
nonionic surfactant Triton X100 .
3) Lipids
for the analysis of high molecular weight lipids like
triacylglycerols.
analyze phospholipids
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. 1) Drugs
 phenothiazine antipscychotics,
 beta blockers,
 felodipine
 clevidipine ,
 methylated betacyclodextrins ,
 vasodialators like isosorbide mononitrate, isosorbide
dinitrate, cyclandelate, nimodipine, amlodipine

29.  oestrogens ,
 combinations of various nonsteroidal antiinflammatory
drugs like flufenamic acid, mefenamic acid, fenbufen,
indomethacin mixtures, flufenamic acid, mefenamic acid,
acetyl salicylic acid, ketoprofen and fenbufen
n Chiral compounds
 , SFC has now become an attractive
alternative for chiral drug separation.

30.  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, diasterioisomers of Du P105- a novel
oxazolidinone antibacterial agent , chiral separation of 1,3 dioxolane
derivatives,
 Organometallics
 Separation of metal chelates and organometals of
thermally labile category, chelates of transition
metals, heavy metals, lanthenides and actinides as
well as organometallic compounds of lead, mercury
and tin has been carried out by SFC. Determination
of solubility of organometallic compounds by SFC
is also reported

31. FLASH CHROMATOGRAPHY
 WHAT IS FLASH CHROMATOGRAPHY ?
 Flash Chromatography is a rapid form of preparative column
chromatography based on optimised pre-packed columns
through which is pumped solvent at a high flow rate. It is a
simple and economical approach to Preparative LC .

32.  After chemical synthesis there is a need to purify the
compounds of interest. The chemist has a choice of techniques
available.
 All of these techniques have a place in the laboratory.
Frequently however, a separation technique is required
because of sample complexity or lack of suitability of the
other techniques

33. These vary in their efficency and resolution. In general, the
higher the resolution, the lower the sample capacity becomes.
For purification, generally the sample amounts are quite high
and therefore flash or column chromatography is the
technique of choice.

34. PRINCIPLE OF FLASH
CHROMATOGRAPHY
 In the traditional column chromatography system ,the user fills
the glass columns with silica gel. This is not desirable because
of serious health concerns regarding breathing in silica gel
dust.
 The sample is placed on the top of the column. Liquid is
passed through the column to elute the sample. Exposure to
organic solvents is not uncommon and not desirable.
 The separation is very slow (typically many hours) and is
restricted to an isocratic solvent mixture.
 At the end of the run, the silica gel must be removed, cleaned,
dried and re-packed. This is both time consuming and
hazardous.

35.  Flash Chromatography
 In the modern Flash Chromatography system the glass
columns are replaced with pre-packed plastic cartridges which
are much safer and also more reproducible.
 Solvent is pumped through the cartridge, which is much
quicker and more reproducible. Systems may also be linked
with detectors and fraction collectors providing automation.
 The introduction of gradient pumps means quicker
separations, less solvent usage and greater flexibility.

36. ADVANTAGES OF FLASH
CHROMATOGRAPHY
 Column Chromatography vs. Flash
Chromatography
 In the example below, an 8 hours glass column
chromatography run was separated in just 50 minutes using a
40mm ID flash cartridge.

37.  Time Savings with Flash Chromatography

38.  Cost Savings with Flash Chromatography
 It would appear that column chromatography is less expensive than flash.
However, flash columns can be re-used multiple times and after taking
labour costs into account, flash chromatography works out significantly
cheaper to run than column chromatography .

39. APPLICATION :
 1) Separating Low Solubility Samples.
 Low solubility samples can cause some difficulties in flash
separation. It is necessary to dissolve them in a solvent matrix
that may not be compatible with the separation solvents.
 In this case, the sample is dissolved in a strong solvent, mixed
with silica and then evaporated to dryness.Placing the
silica/sample mixture in a Solid Sample Introduction Module
(SSIM) allows the sample to be eluted on to the top of the
separation column. Using this technique can improve the
separation dramatically,

40.  2) Separation of Pyridine Derivatives
 3) Separation of polar compounds
 4) Natural Product Purification
 Thus Flash chromatography is an inexpensive and
very useful technique for quickly separating
increasing quantities of samples. It is predictable and
easy to scale up and down as required.
 Modern instrumentation is making it easier still to
take full control over the separation and the technique
continues to develop quickly

41. THANK YOU…