Dr Hanif HPLC

• HPLC


  High performance liquid chromatography - a technique in chromatography that employs high pressure to force liquid or mobile phase into a packed column of stationary phase

  
• Components of an HPLC instrument

  • Solvent
  • Gradient controller and pump
  • Sample Injector
  • Column
  • Detector
  • Data processing system

  
• HPLC column


  • Made from stainless steel tubing which is chemically inert and stable to high pressure
  • Typical length 5-50 cm and inside diameter of 2-6 mm
  • Internal surface should be smooth through the length to prevent irregularities which cause tunneling of mobile phase and band broadening
  • Typical packing range in size from 2-10μm
• Mechanisms of separation in HPLC

  • Partition (normal phase and reverse phase)
  • Adsorption
  • Ion exchange
  • Size exclusion chromatography
  • Affinity chromatography

  
• Normal-phase chromatography


  The stationary phase is highly polar and the mobile phase used is a relatively non-polar solvent such as hexane
• Reversed-phase chromatography


  The stationary phase is non-polar (often a hydrocarbon) and the mobile phase is relatively polar (such as water, methanol or acetonitrile)

  
• About 75% of all HPLC use columns packed with reversed-phase stationary phase