Super Critical Fluid Super Critical Fluid Chromatography Chromatography

Presented by,B.BHAVYA,No. 256213886005,M.Pharm (Pharmaceutics).

Under The Guidence Of Mr. Uttam Prasad M.pharmacy.

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.

DEFINITIO

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DEFINITIO

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• 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.

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.

Contents

• Introduction• Instrumentation:

InjectorsColumnsStationary and Mobile PHASEDetectors

• Advantages • Disadvantages• Comparison • Application• Conclusion

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.

Instrumentation

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

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.

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.

Oven

• A thermostated column oven is required for precise temparature control of the mobile phase. Conventional GC or LC ovens are generally used.

Types of injectorsINJECTOR : 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

Loop injector

In line injector

In column injector

• 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

Columns

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.

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.

• 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.

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.

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.

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.

Disadvantages

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

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.

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).

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.

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

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

• 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.