SUPERCRITICAL FLUID SUPERCRITICAL FLUID CHROMETOGRAPHY AND FLASH CHROMETOGRAPHY AND FLASH

CHROMATOGRAPHY :- PRINCIPLE CHROMATOGRAPHY :- PRINCIPLE AND APPLICATIONAND APPLICATION

PREPARED BY:- ANKIT S. SHAHPREPARED BY:- ANKIT S. SHAH

(09PGPH010)(09PGPH010)

SUPERCRITICAL FLUID CHROMATOGRAPHYSUPERCRITICAL FLUID CHROMATOGRAPHY

ChromatographyChromatography:- separation technique of :- separation technique of complex chemical mixtures into individual complex chemical mixtures into individual components .components .

Various techniques.Various techniques.

Chromatography – mobile phaseChromatography – mobile phase Gas chromatography - GasGas chromatography - Gas Liquid chromatography - LiquidLiquid chromatography - Liquid Supercritical fluid - Supercritical fluid Supercritical fluid - Supercritical fluid chromatography chromatography

S.F.CS.F.C

Cost efficientCost efficient User friendlyUser friendly Better resolutionBetter resolution Faster analysisFaster analysis

S.F.C is a column chromatographic technique in which S.F.C is a column chromatographic technique in which supercritical fluid is used as a mobile phase.supercritical fluid is used as a mobile phase.

What is SUPERCRITICAL FLUID?What is SUPERCRITICAL FLUID? SCF can be described as a fluid obtained by heating above the critical SCF can be described as a fluid obtained by heating above the critical

temperature and compressing above the critical pressuretemperature and compressing above the critical pressure..

Phase Diagram for Pure Substance

For every substance, there is a temperature above which it can For every substance, there is a temperature above which it can no longer exist as a liquid, no matter how much pressure is no longer exist as a liquid, no matter how much pressure is applied.applied.

Likewise, there is a pressure above which the substance can Likewise, there is a pressure above which the substance can no longer exist as a gas no matter how high the temperature is no longer exist as a gas no matter how high the temperature is raised. raised.

These points are called critical temperature and critical These points are called critical temperature and critical pressure respectively. pressure respectively.

Above this point,the substance acts as a supercritical fluid .Above this point,the substance acts as a supercritical fluid .

PROPERTIES AND ADVANTAGES OF SCFPROPERTIES AND ADVANTAGES OF SCF

high densities so they have a remarkable ability to dissolve high densities so they have a remarkable ability to dissolve large, non-volatile molecules .large, non-volatile molecules .

dissolved analytes can be easily recovered by simply allowing dissolved analytes can be easily recovered by simply allowing the solutions to equilibrate with the atmosphere at low the solutions to equilibrate with the atmosphere at low temperatures . So useful with thermally unstable analytes.temperatures . So useful with thermally unstable analytes.

inexpensive inexpensive EcofriendlyEcofriendly

non-toxicnon-toxic

PROPERTIES AND ADVANTAGES OF SCFPROPERTIES AND ADVANTAGES OF SCF

Lower viscosities relative to liquid solventsLower viscosities relative to liquid solvents. . Greater diffusibility means longer column length can be used. Greater diffusibility means longer column length can be used.

Higher diffusion coefficient means higher analysis speed thatHigher diffusion coefficient means higher analysis speed that Comparison of properties of gas,SCF and liquid

Property Property Gas (STP) Gas (STP) SCF SCF Liquid Liquid

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

Diffusion coefficient Diffusion coefficient (cm (cm 2 2 /s) /s)

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

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

The two supercritical fluids of particular interest are , carbon The two supercritical fluids of particular interest are , carbon

dioxide and water.dioxide and water. Carbon dioxideCarbon dioxide :- :- non-flammable, non-flammable, nontoxic nontoxic low critical temperature of 31.9 C and moderate low critical temperature of 31.9 C and moderate

critical pressure of 73bar.critical pressure of 73bar. It is miscible with variety of organic solvents and is It is miscible with variety of organic solvents and is

readily recovered after processing.readily recovered after processing. It diffuses faster than conventional liquid solvents.It diffuses faster than conventional liquid solvents.

Water Water .. critical temperature of 647K and critical pressure of 220bar critical temperature of 647K and critical pressure of 220bar

due to its high polarity.due to its high polarity. The character of water at supercritical conditions changes The character of water at supercritical conditions changes

from one that supports only ionic species at ambient from one that supports only ionic species at ambient conditions to one that dissolves paraffins, aromatics, gases and conditions to one that dissolves paraffins, aromatics, gases and salts.salts.

Due to this unique property, research has been carried out on Due to this unique property, research has been carried out on supercritical water for reaction and separation processes to supercritical water for reaction and separation processes to treat toxic wastewater.treat toxic wastewater.

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

PRINCIPLEPRINCIPLE

INSTRUMENTATIONINSTRUMENTATION

The instrumentation of SFC is similar in most regards to The instrumentation of SFC is similar in most regards to instrumentation for HPLC because the pressure and instrumentation for HPLC because the pressure and temperature required for creating supercritical fluid from temperature required for creating supercritical fluid from several gases or liquids lie well within the operating limits of several gases or liquids lie well within the operating limits of

HPLC equipmentHPLC equipment However, there are two main differences between the two.However, there are two main differences between the two. 1.1. a thermostated oven required to provide precise temperature a thermostated oven required to provide precise temperature

control of the mobile phasecontrol of the mobile phase 2.2. a restrictor to maintain the pressure in the column at a a restrictor to maintain the pressure in the column at a

desired level and to convert the eluent from SCF to a gas for desired level and to convert the eluent from SCF to a gas for

transfer to detectortransfer to detector . .

Flow Diagram of Construction of SFC Instrument

the mobile phase is pumped as a liquid and is brought into the the mobile phase is pumped as a liquid and is brought into the supercritical region by heating it above its supercritical supercritical region by heating it above its supercritical temperature before it enters the analytical column. temperature before it enters the analytical column.

It passes through an injection valve where the sample is It passes through an injection valve where the sample is introduced into the supercritical stream introduced into the supercritical stream

It is maintained supercritical as it passes through the column It is maintained supercritical as it passes through the column into the detector by a pressure restrictor. into the detector by a pressure restrictor.

Mobile phase:-Mobile phase:- There are a number of possible fluids, which may be used in There are a number of possible fluids, which may be used in

SFC as a mobile phaseSFC as a mobile phase.. However, based on its low cost, low interference with However, based on its low cost, low interference with

chromatographic detectors and good physical properties chromatographic detectors and good physical properties (nontoxic, nonflammable, low critical values) CO(nontoxic, nonflammable, low critical values) CO2 2 is the most is the most

used mobile phase for SFCused mobile phase for SFC . . excellent solvent for a variety of nonpolar organic molecules.excellent solvent for a variety of nonpolar organic molecules.

ColumnsColumns Basically two types of analytical columns are used in SFC, Basically two types of analytical columns are used in SFC,

packed and capillary.packed and capillary. Earlier work employed absorbents such as alumna, silica or Earlier work employed absorbents such as alumna, silica or

polystyrene .polystyrene . More recent packed column work has involved bonded More recent packed column work has involved bonded

stationary phases such as octadecylsilyl (C 18 ).stationary phases such as octadecylsilyl (C 18 ). OvenOven A thermostated column oven is required for precise A thermostated column oven is required for precise

temperature control of the mobile phasetemperature control of the mobile phase. .

Restrictor Restrictor This is a device, which is used to maintain desired pressure in This is a device, which is used to maintain desired pressure in

the column by the column by - a pressure-adjustable diaphragm or- a pressure-adjustable diaphragm or - controlled nozzle - controlled nozzle so that the same column-outlet pressure is maintained so that the same column-outlet pressure is maintained

irrespective of the mobile phase pump flow rate.irrespective of the mobile phase pump flow rate. It keeps the mobile phase supercritical throughout the It keeps the mobile phase supercritical throughout the

separation and often must be heated to prevent clogging. separation and often must be heated to prevent clogging. The pressure restrictor is placed either after the detector or at The pressure restrictor is placed either after the detector or at

the end of the column.  the end of the column. 

MicroprocessorMicroprocessor The commercial instruments for SFC are ordinarily equipped The commercial instruments for SFC are ordinarily equipped

with one or more microprocessors to control such variables as with one or more microprocessors to control such variables as pumping pressures, oven temperature and detector pumping pressures, oven temperature and detector

performanceperformance. . DetectorDetector

it is compatible with both HPLC and GC detectors.it is compatible with both HPLC and GC detectors. flame photometric detectors flame photometric detectors

flame ionization detectorsflame ionization detectors

refractive index detectors refractive index detectors ultraviolet-visible spectrophotometric detectors ultraviolet-visible spectrophotometric detectors light scattering detectors light scattering detectors The choice of detectors will depend upon the mobile phase The choice of detectors will depend upon the mobile phase

composition, column type, flow rate and ability to withstand composition, column type, flow rate and ability to withstand

the high pressures of SFC.the high pressures of SFC.

Modifiers Modifiers CO 2  is not a very good solvent for high molecular weight, CO 2  is not a very good solvent for high molecular weight,

ionic and polar analytes ionic and polar analytes This can be overcome by adding a small portion of a second This can be overcome by adding a small portion of a second

fluid called modifier fluid fluid called modifier fluid This is generally an organic solvent, which is completely This is generally an organic solvent, which is completely

miscible with carbon dioxidemiscible with carbon dioxide methanol, acetonitrile, ethanol and 1-propanol.methanol, acetonitrile, ethanol and 1-propanol.

COMPARISION WITH OTHER TYPES OF COMPARISION WITH OTHER TYPES OF

CHROMATOGRAPHY:-CHROMATOGRAPHY:- Several physical properties of SCF are intermediate between Several physical properties of SCF are intermediate between

gases and liquids.gases and liquids. SFC is inherently faster than LC because the lower viscosity SFC is inherently faster than LC because the lower viscosity

makes use of higher flow rates.makes use of higher flow rates. ability to separate thermally labile compounds (20% drugs)ability to separate thermally labile compounds (20% drugs) SFC is faster than HPLC, because of its lower viscosity and SFC is faster than HPLC, because of its lower viscosity and

higher diffusion rateshigher diffusion rates

Unlike GC, by changing the mobile phase the selectivity can Unlike GC, by changing the mobile phase the selectivity can be varied in SFC . be varied in SFC .

Due to the thermally unstable or non- –volatile nature of many Due to the thermally unstable or non- –volatile nature of many nitrogen and / or sulfur containing compounds, they cannot be nitrogen and / or sulfur containing compounds, they cannot be analyzed by GC . Because SFC generally uses carbon dioxide, analyzed by GC . Because SFC generally uses carbon dioxide, collected as a byproduct of other chemical reactions or is collected as a byproduct of other chemical reactions or is collected directly from the atmosphere, it contributes no new collected directly from the atmosphere, it contributes no new chemicals to the environmentchemicals to the environment..

biggest advantage that SFC has over HPLC lies within the biggest advantage that SFC has over HPLC lies within the differences in the mobile phases.differences in the mobile phases.

Supercritical fluids are less viscous, possess a higher Supercritical fluids are less viscous, possess a higher diffusivity than liquids under HPLC conditions.This provides diffusivity than liquids under HPLC conditions.This provides not only the ability to increase column lengths, but also allows not only the ability to increase column lengths, but also allows for faster flow rates. for faster flow rates.

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

been key in many chiral separationsbeen key in many chiral separations . .

APPLICATION OF SFCAPPLICATION OF SFC

By now SFC has been applied to wide variety of By now SFC has been applied to wide variety of materials. materials.

natural products, natural products, drugs,drugs, foods,foods, pesticides, pesticides, herbicides,herbicides, surfactants,surfactants, polymers and polymer additives, polymers and polymer additives, Chiral compoundChiral compound

1)1) Natural ProductsNatural Products Lipophilic – amphiphilic compounds with properties Lipophilic – amphiphilic compounds with properties

between volatiles and hydrophilic compounds often create between volatiles and hydrophilic compounds often create problems in connection with their isolation and analytical problems in connection with their isolation and analytical determination resulting in an analytical gray area,determination resulting in an analytical gray area,

But SFC has been found to give relatively fast and simple But SFC has been found to give relatively fast and simple procedures for determination of oil constituents such as procedures for determination of oil constituents such as chlorophyll and its derivatives .chlorophyll and its derivatives .

Separation of bile salts and common free bile acids like Separation of bile salts and common free bile acids like ursodeoxycholic acid and chenodeoxycholic acid in ursodeoxycholic acid and chenodeoxycholic acid in pharmaceutical preparation.pharmaceutical preparation.

separation of underivatized triterpene acids separation of underivatized triterpene acids estimation of caffeine from tea and conjugated bile acids estimation of caffeine from tea and conjugated bile acids analysis of panaxadiol / panaxatriol in ginseng .analysis of panaxadiol / panaxatriol in ginseng .

2) Pesticides2) Pesticides

analysis of pesticide residues in canned foods, fruits and analysis of pesticide residues in canned foods, fruits and vegetables wherein pyrethroids, herbicides, fungicides and vegetables wherein pyrethroids, herbicides, fungicides and carbamates have been tested .carbamates have been tested .

1)1) SurfactantsSurfactants Separation of the oligomers in a sample of the Separation of the oligomers in a sample of the

nonionic surfactant Triton X100 .nonionic surfactant Triton X100 .1)1) Lipids Lipids for the analysis of high molecular weight lipids like for the analysis of high molecular weight lipids like

triacylglycerols. triacylglycerols. analyze phospholipids analyze phospholipids Separation of fatty acid methyl esters , biosynthetic Separation of fatty acid methyl esters , biosynthetic

polyunsaturated fatty acids (PUFA) 37 , polyunsaturated fatty acids (PUFA) 37 , nonsaponifiable lipids , cholesterol and its esters in nonsaponifiable lipids , cholesterol and its esters in human serum and food sampleshuman serum and food samples

1)1) Drugs Drugs phenothiazine antipscychotics,phenothiazine antipscychotics, beta blockers, beta blockers, felodipine felodipine clevidipine , clevidipine , methylated betacyclodextrins , methylated betacyclodextrins , vasodialators like isosorbide mononitrate, isosorbide vasodialators like isosorbide mononitrate, isosorbide

dinitrate, cyclandelate, nimodipine, amlodipine dinitrate, cyclandelate, nimodipine, amlodipine

oestrogens , oestrogens , combinations of various nonsteroidal antiinflammatory drugs combinations of various nonsteroidal antiinflammatory drugs

like flufenamic acid, mefenamic acid, fenbufen, indomethacin like flufenamic acid, mefenamic acid, fenbufen, indomethacin mixtures, flufenamic acid, mefenamic acid, acetyl salicylic mixtures, flufenamic acid, mefenamic acid, acetyl salicylic acid, ketoprofen and fenbufen acid, ketoprofen and fenbufen

1)1) Chiral compoundsChiral compounds

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

SFC has been applied to separation of a large number of enantiomers, SFC has been applied to separation of a large number of enantiomers, diasterioisomers and geometrical isomers like achiral and chiral analysis diasterioisomers and geometrical isomers like achiral and chiral analysis of camazepam and its metabolites, diasterioisomers of Du P105- a novel of camazepam and its metabolites, diasterioisomers of Du P105- a novel oxazolidinone antibacterial agent , chiral separation of 1,3 dioxolane oxazolidinone antibacterial agent , chiral separation of 1,3 dioxolane derivatives,derivatives,

Organometallics Organometallics Separation of metal chelates and organometals of Separation of metal chelates and organometals of

thermally labile category, chelates of transition thermally labile category, chelates of transition metals, heavy metals, lanthenides and actinides as metals, heavy metals, lanthenides and actinides as well as organometallic compounds of lead, mercury well as organometallic compounds of lead, mercury and tin has been carried out by SFC. Determination and tin has been carried out by SFC. Determination of solubility of organometallic compounds by SFC of solubility of organometallic compounds by SFC is also reported is also reported

FLASH CHROMATOGRAPHYFLASH CHROMATOGRAPHY

WHAT IS FLASH CHROMATOGRAPHY ?WHAT IS FLASH CHROMATOGRAPHY ? Flash Chromatography is a rapid form of preparative column Flash Chromatography is a rapid form of preparative column

chromatography based on optimised pre-packed columns chromatography based on optimised pre-packed columns through which is pumped solvent at a high flow rate. It is a through which is pumped solvent at a high flow rate. It is a simple and economical approach to Preparative LC .simple and economical approach to Preparative LC .

After chemical synthesis there is a need to purify the After chemical synthesis there is a need to purify the compounds of interest. The chemist has a choice of techniques compounds of interest. The chemist has a choice of techniques available.available.

All of these techniques have a place in the laboratory. All of these techniques have a place in the laboratory. Frequently however, a separation technique is required Frequently however, a separation technique is required because of sample complexity or lack of suitability of the because of sample complexity or lack of suitability of the other techniquesother techniques

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.

PRINCIPLE OF FLASH PRINCIPLE OF FLASH CHROMATOGRAPHYCHROMATOGRAPHY

In the traditional column chromatography system ,the user fills In the traditional column chromatography system ,the user fills the glass columns with silica gel. This is not desirable because the glass columns with silica gel. This is not desirable because of serious health concerns regarding breathing in silica gel of serious health concerns regarding breathing in silica gel dust.dust.

The sample is placed on the top of the column. Liquid is The sample is placed on the top of the column. Liquid is passed through the column to elute the sample. Exposure to passed through the column to elute the sample. Exposure to organic solvents is not uncommon and not desirable.organic solvents is not uncommon and not desirable.

The separation is very slow (typically many hours) and is The separation is very slow (typically many hours) and is restricted to an isocratic solvent mixture.restricted to an isocratic solvent mixture.

At the end of the run, the silica gel must be removed, cleaned, At the end of the run, the silica gel must be removed, cleaned, dried and re-packed. This is both time consuming and dried and re-packed. This is both time consuming and hazardous.hazardous.

Flash ChromatographyFlash Chromatography In the modern Flash Chromatography system the glass In the modern Flash Chromatography system the glass

columns are replaced with pre-packed plastic cartridges which columns are replaced with pre-packed plastic cartridges which are much safer and also more reproducible.are much safer and also more reproducible.

Solvent is pumped through the cartridge, which is much Solvent is pumped through the cartridge, which is much quicker and more reproducible. Systems may also be linked quicker and more reproducible. Systems may also be linked with detectors and fraction collectors providing automation.with detectors and fraction collectors providing automation.

The introduction of gradient pumps means quicker The introduction of gradient pumps means quicker separations, less solvent usage and greater flexibility.separations, less solvent usage and greater flexibility.

ADVANTAGES OF FLASH ADVANTAGES OF FLASH

CHROMATOGRAPHYCHROMATOGRAPHY Column Chromatography vs. Flash Column Chromatography vs. Flash

ChromatographyChromatography In the example below, an 8 hours glass column In the example below, an 8 hours glass column

chromatography run was separated in just 50 minutes using a chromatography run was separated in just 50 minutes using a 40mm ID flash cartridge. 40mm ID flash cartridge.

Time Savings with Flash ChromatographyTime Savings with Flash Chromatography

Cost Savings with Flash ChromatographyCost Savings with Flash Chromatography It would appear that column chromatography is less expensive than flash. It would appear that column chromatography is less expensive than flash.

However, flash columns can be re-used multiple times and after taking However, flash columns can be re-used multiple times and after taking labour costs into account, flash chromatography works out significantly labour costs into account, flash chromatography works out significantly cheaper to run than column chromatography .cheaper to run than column chromatography .

APPLICATION :APPLICATION : 1) Separating Low Solubility Samples.1) Separating Low Solubility Samples. Low solubility samples can cause some difficulties in flash Low solubility samples can cause some difficulties in flash

separation. It is necessary to dissolve them in a solvent matrix separation. It is necessary to dissolve them in a solvent matrix that may not be compatible with the separation solvents.that may not be compatible with the separation solvents.

In this case, the sample is dissolved in a strong solvent, mixed In this case, the sample is dissolved in a strong solvent, mixed with silica and then evaporated to dryness.Placing the with silica and then evaporated to dryness.Placing the silica/sample mixture in a Solid Sample Introduction Module silica/sample mixture in a Solid Sample Introduction Module (SSIM) allows the sample to be eluted on to the top of the (SSIM) allows the sample to be eluted on to the top of the separation column. Using this technique can improve the separation column. Using this technique can improve the separation dramatically,separation dramatically,

2) Separation of Pyridine Derivatives2) Separation of Pyridine Derivatives 3) Separation of polar compounds3) Separation of polar compounds 4) Natural Product Purification4) Natural Product Purification

ThusThus Flash chromatography Flash chromatography is an inexpensive and is an inexpensive and very useful technique for quickly separating very useful technique for quickly separating increasing quantities of samples. It is predictable and increasing quantities of samples. It is predictable and easy to scale up and down as required.easy to scale up and down as required.

Modern instrumentation is making it easier still to Modern instrumentation is making it easier still to take full control over the separation and the technique take full control over the separation and the technique continues to develop quicklycontinues to develop quickly

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