Prepared by :

SHINDE GANESH SHASHIKANTPRAVARA RURAL COLLEGE OF PHARMACY,PRAVARANAGAR

SUPERCRITICAL FLUID

CHROMETOGRAPHY

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

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.

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

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 .

A supercritical fluid is any substance at a

temperature and pressure above its critical point.

It can diffuse through solids like a gas, and dissolve

materials like a liquid.

Additionally, close to the critical point, small

changes in pressure or temperature result in large

changes in density, allowing many properties to be

"tuned".

Supercritical fluids are suitable as a substitute for

organic solvents in a range of industrial and

laboratory processes

In the Supercritical region the substance is neither a gas nor

a liquid – it is a fluid that has properties of both.

There are no sharp boundaries between gas and liquid.

Properties of SCFs can be very different from the normal

liquid phase.

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

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

Property

Gas (STP) SCF Liquid

Density (g/cm 3 ) (0.6-2) x 10 -3 0.2-0.5 0.6-2

Diffusion coefficient

(cm 2 /s)

(1-4) x 10 -1 10 -3 x 10 - 4 (0.2-2) x 10 -5

Viscosity (G Cm -1 s -1 ) (1-4) x 10 - 4 (1-3) x 10 - 4 (0.2-3) x 10 -2

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.

Carbon dioxide pressure-temperature phase diagram

Above the critical point, the

phase boundary (meniscus)

between liquid and vapor

phases disappears, and the

substance is a single

homogeneous fluid

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.

Water Pressure-temperature Phase Diagram

Density

(kg/m3)

Viscosity

(µPa∙s)

Diffusivity

(mm²/s)

Gases 1 10 1-10

Supercritical

Fluids

100-1000 50-100 0.01-0.1

Liquids 1000 500-1000 0.001

Comparison of Gases, Supercritical Fluids and Liquids

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.

1. a thermostated oven required to provide precise temperature

control of the mobile phase

2. 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 .

Supercritical fluid chromatography

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.

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.

Column

Basically two types of analytical columns are used in SFC,

1) packed column

2) capillary column.

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 ).

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.

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

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.

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.

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 .

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.

SFC can be set up for sub ambient temperatures, which has

been key in many chiral separations .

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

1) 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.

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 .

1) Surfactants

Separation of the oligomers in a sample of the nonionic surfactant Triton X100 .

1) Lipids

for the analysis of high molecular weight lipids liketriacylglycerols.

analyze phospholipids

Separation of fatty acid methyl esters , biosyntheticpolyunsaturated fatty acids (PUFA) 37 ,nonsaponifiable lipids , cholesterol and its esters inhuman serum and food samples

1) Drugs 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, flufenamic acid, mefenamic acid,

acetyl salicylic acid, ketoprofen and fenbufen

1) Chiral compounds

, SFC has now become an attractive alternative for chiral drug separation.

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

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