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HPLC

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HPLC

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    1. HPLC High Performance/Pressure Liquid Chromatography

    2. Advanced Instrumentation

    3. The System

    4. Columns

    6. Reversed Phase Chromatography Packing is nonpolar Solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase - hydrophobic.

    7. Reversed Phase Chromatography - 2 Bonded phases made by covalently bonding a molecule onto a solid stationary phase like silica Typical stationary phases are nonpolar hydrocarbons, waxy liquids or bonded hydrocarbons (such as C18, C8, C4, etc.) pH range 2.5 to 7.5 ONLY (or the column degrades) 60-90% of all analytical LC separations are done on bonded phases in the reverse phase mode.

    8. Reversed Phase Chromatography - 3 Solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water. Elution can be either: Isocratic use only one buffer Gradient mix in a second buffer

    9. Capacity Analytical: ng down to fg Semi-prep: mg to ug Preparative: g Industrial: kg

    10. Solvents Clean HPLC grade Filtered Degassed Water Polar Methanol or Acetonitrile Maintain pH TFA

    11. Tubing Very small inner diameter Consistent i.d. Very strong Easy to cut Fittings available

    12. Injector

    13. Pumps

    14. Pump schematic

    15. Pump cycle

    16. Detector

    17. Detector schematic

    18. Fraction collector

    19. Recorder

    20. Direct to computer

    21. HPLC Data

    22. HPLC Data

    23. HPLC Data Analysis

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