Supercritical chromatography

SUPERCRITICAL CHROMATOGRAPHY USES SUPERCRITICAL FLUID AS A MOBILE PHASE AND USE FOR ANALYSIS OF PHARMACEUTICAL AND CRUDE DRUGS AND CHEMICALS.

1. Prepared by : SHINDE GANESH SHASHIKANT PRAVARA RURAL COLLEGE OF PHARMACY,PRAVARANAGAR SUPERCRITICAL FLUID CHROMETOGRAPHY

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 point s are called critical temperature and critical pressure respectively. Above this point,the substance acts as a supercritical fluid .

6. 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

7. 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 .

8. 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

9. 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

10. 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.

11. Carbon dioxide pressure-temperature phase diagram

12. Above the critical point, the phase boundary (meniscus) between liquid and vapor phases disappears, and the substance is a single homogeneous fluid

13. 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.

14. Water Pressure-temperature Phase Diagram

15. Density (kg/m 3 ) Viscosity ( µ Pa∙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

16. 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 .

17. Supercritical fluid chromatography

18. 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.

19. 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) CO 2 is the most used mobile phase for SFC . excellent solvent for a variety of nonpolar organic molecules.

20. C olumn 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 ).

21. 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. 

22. 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 .

23. Detector I t 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.

24. 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.

25. 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 .

26. 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.

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

28. 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

29. 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.

30. 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 .

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

32. Drugs phenothiazine antipscychotics, beta blockers, felodipine clevidipine , methylated betacyclodextrins , vasodialators like isosorbide mononitrate, isosorbide dinitrate, cyclandelate, nimodipine, amlodipine

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

34. 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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