Super critical fluid chromatography

Super critical fluid, Instrumentation

1. SUPER CRITICAL FLUID CHROMATOGRAPHY PREPARED BY: DEVIPRIYA P V M PHARM DEPT OF PHARMACEUTICAL ANALYSIS

2. INTRODUCTION SFC is a separation technique in which the 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 analyte(s) and stationary phase in the column. Hybrid of Gas chromatography (GC)and HPLC 2

3. CRITICAL TEMPERATURE: The temperature above which a distinct liquid phase cannot exist, regardless of pressure. CRITICAL PRESSURE: The vapour pressure of a substance at its critical temperature 3

4. Super critical fluid A supercritical fluid is formed whenever a substance is heated above its critical temperature. Have diffusion coefficient close to those of gases. Low viscosity. No surface tension properties(absence of surface meniscus). 4

5. Properties of some supercritical fluids Fluid Critical Temperature Critical Pressure Critical point g/ml Density at 400atm, g/ml CO 2 31.3 72.9 0.47 0.96 N 2 O 36.5 71.7 0.45 0.94 NH 3 132.5 112.5 0.24 0.40 n-Butane 152.0 37.5 0.23 0.50 5

6. CO 2 as super critical fluid Critical temperature(31°C)is only slightly above room temperature. The critical pressure of CO 2 (73.8 bar) is not difficult to maintain. Non flammable. Chemically inert. Odour free. Available in a state of high purity at low cost. No solvent disposal problem. Considered green (recycled). 6

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8. INSTRUMENTATION Mobile phase Stationary phase Pumps. Injectors. Oven. SFC column. Detector. Restrictor. 8

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10. PUMPS For flow controlling. Syringe pumps are used for capillary SFC for consistent pressure. For packed columns for easier blending of the mobile phase or introduction of modifier fluids reciprocating pumps are used. 10

11. OVEN Thermostatted column oven For precise temperature control of the mobile phase. Conventional GC or LC ovens are generally used. 11

12. BACK PRESSURE DEVICE- A RESTRICTOR Used to maintain the desired pressure in the column. To convert the eluent from a supercritical fluid to a gas for transfer to the detector. Keeps the mobile phase super critical throughout separation and often must be heated to prevent clogging. Placed either at the end of the column or after detector. 12

13. INJECTORS In capillary SFC small sample should be quickly injected into the column so pneumatically driven valves are used. For packed SFC a typical injection valve is commonly used. Types of injectors: Loop injector In column injector. Inline injector. 13

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15. COLUMNS Once the sample is injected into the super critical stream, it is carried into the analytical column that contains a highly viscous liquid (stationary phase) into which the analyte can be temporarily adsorbed and then released based on their chemical nature. 15

16. Two types: Packed columns: contains small deactivated particles so that the stationary phase adheres the column are conventionally stainless steel. Capillary columns: are open tubular columns of narrow internal diameter made of fused silica with the stationary phase bonded to the wall of the column. 16

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18. STATIONARY PHASES Silica/Alumina: Useful for non polar compounds. Lead to irreversible adsorption of some polar solutes. Bonded to provide less adsorptive packing material. Need organic modifiers to elute analytes . 18

19. Widely used polar stationary phase: Polysiloxane : stable, flexible , Si-O bond leads to good diffusion. Poly methyl siloxanes : increase efficiency in separating closely related analytes . 19

20. MOBILE PHASE Most widely used mobile phase for SFC is CO2. It is an excellent solvent for a variety of non polar organic molecules. It transmits in the UV region and is odourless , non toxic, readily available, and remarkably inexpensive. 20

21. Very polar or ionic compounds are not able to be eluted. This can be overcome by adding polar organic modifiers, such as methanol. Improves solvating ability of supercritical fluid and sometimes enhances selectivity of separation efficiency by blocking some of the highly active sites on the stationary phase. Modifiers are commonly used in packed columns . 21

22. DETECTOR Flame Ionization Detector 22

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24. 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. Rapid separation without use of organic solvents. Uses environmentally conscious technology. High resolution at lower temperature. High diffusion coefficient. Low viscosity 24

25. DISADVANTAGES Elevated pressure required. Relative high cost of equipment. unusual operating conditions. Complicated phase behaviour. Expensive technology. Cleaning will be time consuming. 25

26. APPLICATIONS Chiral separation of the enantiomers of a molecule. Purification of each of the enantiomers in sufficient quantities to permit a study of the enantiomer’s pharmacokinetic and metabolic properties. Identification of the enantiomer of choice as a possible therapeutic agent. Purification on higher (production) scales. 26

27. Analysis of complex oligomeric mixtures including polypropylene glycol, Polysiloxane, fluorocarbon oligomers and high molecular weight normal alcohols. : Mobile phase :CO 2 at 140°C. : Fused silica capillary coated with a 0.25 μ m film of 5% polysiloxane . Separation of mono-, di-, and triglycerides at low operating. separations of complex hydrocarbon mixtures from liquid fuels, polycyclic aromatic hydrocarbons, synthetic alpha-olefins, and petroleum functional groups. :Mobile phase : pentane at 210°C : Capillary coated with 0.25 μ m film of 50% phenyl polysiloxane . 27

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