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Supercritical Fluid Chromatography

Supercritical Fluid Chromatography. Aurelia Leyva and Trang Duong Chem 230 11/30/2010. Outline. Background Apparatus Injectors Column Mobile & Stationary Phase Detectors Advantages Disadvantages Comparison Applications Conclusions. Supercritical Fluid. T/P Phase Diagram.

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Supercritical Fluid Chromatography

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  1. Supercritical Fluid Chromatography Aurelia Leyva and Trang Duong Chem 230 11/30/2010

  2. Outline • Background • Apparatus • Injectors • Column • Mobile & Stationary Phase • Detectors • Advantages • Disadvantages • Comparison • Applications • Conclusions

  3. Supercritical Fluid T/P Phase Diagram • Substance at a T and P above critical point • Effuse through solids like a gas and dissolve materials like a liquid • Close to critical point, small changes in T or P results in large changes in density www.che.tohoku.ac.jp W.W. Christie and Lipid Technology

  4. Supercritical Fluid Chromatography (SFC) • Hewlett-Packard developed and manufactured the SFC instrument in 1982 • Combinations of desirable characteristics from both GC and HPLC • Used for compounds: • Highly polar • Large MW • Too thermally labile for GC • Compounds undetectable for HPLC detector Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011

  5. SFC Apparatus 1. Mobile phase (CO2) 5. Column 2. Pump 6. Detector 3. Injection System 7.Chromatogram 4. Oven http://www.cee.vt.edu/ewr/environmental/teach/smprimer/sfc/sfc.html

  6. Types of Injectors • Loop injection • -Low pressure feed pump needed to fill • the loop • -Mostly for preliminary tests of column • performance and elution parameters • b.In-line injection • -Flexibility for changing injected volume • -High-pressure pump required to inject • feed solution • -Injected stream dissolved in eluent flow • c.In-column injection • -Permits injection of the feed solution • directly onto the column • -No dilutions required 6 Journal of Liquid Chromatography & Related Technologies, 28: 1233–1252, 2005

  7. Injector Volumes • Open tubular columns • Injection volumes >96nL • Greater volume affects resolution • Packed columns • Injection volumes >1uL

  8. Column • Open Tubular • Smaller column diameter more efficient • - Efficiency decreases at high density (decrease u) • * Decrease column diameter • * Increase T (increase diffusion, solubility, volatility) • - Minimal pressure drop effect J.W. King, H.H. Hill, and M.L. Lee. 1993 8

  9. Column • Packed • Packing reduces permeability and creates flow resistance, L <25cm • Efficiency increases with increasing density (Dm decreases) • Smaller particle size = smaller H, but increase pressure drop, and decrease permeability • Large P drop effect (density decreases, solubility decreases along column length) J.W. King, H.H. Hill, and M.L. Lee. 1993

  10. Column Retention Time -Packed columns are more efficient with higher density analytes. • Packed capillary • Lower volumetric flow rate = more compatible with mass flow detector, greater sensitivity in conc. detector, easier sample transfer in multi-D system, higher permeability-longer column J.W. King, H.H. Hill, and M.L. Lee. 1993

  11. Stationary Phase • Same as those for GC and LC, with some modification. • Silica/Alumina • Useful for non-polar compounds • Lead to irreversible adsorption of some polar solutes • Bonded to provide less adsorptive packing material • Octyl, octadecyl, cyanoalkyl, aminoalkyl, diolakyl • Need organic modifiers to elute analytes • Widely used polar S Phase • Polysiloxanes-- stable, flexible Si--O bond lead to good diffusion. • Substituted with chemical groups for selective interaction with analyte • Polymethylsiloxanes--increase efficiency in separating closely related polar analytes • Cyanopropyl polysiloxanes-useful for compounds with –COOH

  12. Mobile Phase Neat Fluid • CO2 low Tc, low toxicity, non-flammability • N2O high degree of nonideality, permits high fluid density when compressed • Isopropyl/diethyl ether separation of polynuclear aromatics and polymers. • Hydrocarbon limited use due to high flammability • n-Pentane separation of oligomers • Flourocarbon low Tc, unique selectivity for certain solutes 12 J.W. King, H.H. Hill, and M.L. Lee. 1993

  13. Mobile Phase Mix Fluid: Addition of polar organic modifier -Enhance solubility of polar analyte -Reduce retention volume -Eliminate strong interaction between absorptive site and polar solute -Will modify polarity of eluent (asso. with ε and polarity index) -k and α affected by modifier identity and concentration Caution when using mixed fluids to assure that the components are miscible over the range of T and P used.

  14. Detectors Can use either GC or LC detectors with adaptations -LC detector: in close cell, fluid maintain under pressure, but cooled liquid -GC detector: in open cell, fluid decompressed to gas Ambient Pressure Ionization FID : organics hydrogen-atmosphere FID: organometallics thermionic detector: N, P containing compounds ECD: Halogenated/electronegative compounds photoionization detector: organics with less than 10.2eV ionization potential ion mobility detector: high electron/proton affinity compounds Optical UVDFT-IR Flourescence detector FPD (flame photometric detector): S, P containing compounds CD (chemiluminescence detector): S, and easily oxidizable compounds Element selective plasma emission detector LSD (light scattering detector) Vacuum MS with (CI, EI, CE, API ionizer)

  15. SFC Comparison Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011

  16. SFC Comparison J.W. King, H.H. Hill, and M.L. Lee. 1993

  17. SFC Advantages • Provides rapid separations without the use of organic solvents • Uses environmentally conscious technology • Faster separation process • Decrease in resistance to mass transfer in column • High resolution at lower temperature • Low viscosity • High diffusion coefficient • Greater diffusibility Hemaxi S. Bhatt et al. / Journal of Pharmacy Research 2009, 2(10),1606-16011

  18. SFC Disadvantages • SCF is a recent chromatography technique that requires development in the following areas: • Method development • Hardware development • Pump design • Injection methodology • Expensive technology • Pressure & Temperature • Cleaning is time consuming Ind. Eng. Chem. Res. 2000, 39, 4531-4535

  19. Applications • Fossil Fuels and Hydrocarbons • Agrichemicals • Drugs and Pharmaceuticals • Polymers • Explosives and Propellants • Lipids • Carbohydrates • Industrial Chemicals • Foods and Flavors • Natural Products • Metal Chelates and Organometallic Compounds • Enantiomers

  20. Agrichemical Application Chrysene • Pesticide detection • GC conditions: • 15m x 200pm • Open tubular column • 0.15 q film thickness • Temp. program from 40 to 200°C at l0°C/min, then 4°C/min to 240°C • SFC conditions: • 10m x 50pm • Open tubular column • 0.15 pm film thickness • CO2 100°C • Density program from 0.55 to 0.70g/mL at 0.005g/mL per min after a 10-min GC SFC J. W. KING, H. H. HILL, AND M. L. LEE

  21. Natural Products Application • Cholesterol content of a fish oil • Content of capsule was emptied into injection solvent n-hexane • The oil-laden solution was directly injected into SFC & separated using density program • Two peaks were eluted with in program density interval 0.40-0.42 g/mL • Found by spiking the above solution with known amounts of alpha-tocopherol (vitamin E) and cholesterol Journal of Chromatographic Science, Vol. 28, January 1990

  22. References • M. Perrut. Supercritical Fluid Applications: Industrial Developments and Economic Issues. Ind. Eng. Chem. Res., 39, 12, 2000 • J. W. King. Applications of Capillary Supercritical Fluid Chromatography-Supercritical Fluid Extractions to Natural Products.Journal of Chromatographic Science, 28, 1990 • W. Majewski, E. Valery, O. Ludemann-Hombourger. Principle and Applications of Supercritical Fluid Chromatography. Journal of Liquid Chromatography & Related Technologies, 28, 1233–1252, 2005 • H. S. Bhatt, G. F. Patel, N. V. Vekariya1, S. K. Jadav. Super critical fluid chromatography-an overview. Journal of Pharmacy Research, 2(10),1606-16011, 2009 • J. W. King, H. H. Hill, M. L. Lee. Analytical Supercritical Fluid Chromatography and Extraction. Ind. Eng. Chem. Res., Vol. 39, No. 12, 1993

  23. Capillary SFC • capillary SFC can be used to separate the analytes of interest from a relatively nonvolatile sample matrix without resorting to sample preparation prior to chromatography. • capillary SFC include the separation of reaction products from higher molecular weight starting materials and the deformulation of commercial products. • Capillary SFC, when coupled with micro-scale SFE also permits the characterization of small samples, such as portions of single seeds and extractables from single, live insects. 23 Journal of Chromatographic Science, Vol. 28, January, 1990

  24. PROBLEMS • What controls the pressure in SFC? • The restrictor • Why is a modifier fluid needed? • To help elute polar analytes • List one advantage of SFC compared to HPLC and GC? • can analyze high MW, non-volatile compound, higher resolution compare to GC (see slide 17) • Compare the plate number (N) between an open tubular (Ds = 1x10-6cm2/s, dc = 50um, df = 0.25um, L = 10m, u = 5.8cm/s) and packed column (ko = 0.5, dp = 5um, L = 0.1m, u = 2.1cm/s). k=2, and Dm = 2x10-4cm2/s. • OT (N) = 19000, Packed (N)= 9100

  25. Important Info Web pages • http://www.google.com/imgres?imgurl=http://www.chem.leeds.ac.uk/People/CMR/images/scco24.jpg&imgrefurl=http://www.chem.leeds.ac.uk/People/CMR/criticalpics.html&usg=__y5nA5mu0N3cnYx3-cqlUrb7dKik=&h=294&w=308&sz=54&hl=en&start=3&zoom=1&um=1&itbs=1&tbnid=chFV9_BtWriMMM:&tbnh=112&tbnw=117&prev=/images%3Fq%3Dsupercritical%2Bfluid%26um%3D1%26hl%3Den%26sa%3DN%26tbs%3Disch:1 • http://en.wikipedia.org/wiki/Supercritical_fluid

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