SUPERCRITICAL FLUIDCHROMETOGRAPHY AND FLASHCHROMATOGRAPHY :- PRINCIPLE AND APPLICATION PREPARED BY:- ANKIT S. SHAH (09PGPH010)
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 .
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 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
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.
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.
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.
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 .
Flow Diagram of Construction of SFC Instrument
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.
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.
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
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.
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 .
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
oestrogens , combinations of various nonsteroidal antiinflammatory drugs like flufenamic acid, mefenamic acid, fenbufen, indomethacin mixtures, flufenamic acid, mefenamic acid, acetyl salicylic acid, ketoprofen and fenbufenn 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
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 .
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
These vary in their efficency and resolution. In general, thehigher the resolution, the lower the sample capacity becomes.For purification, generally the sample amounts are quite highand therefore flash or column chromatography is thetechnique of choice.
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.
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.
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.
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 .
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,
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