High Performance Liquid Chromatography. HPLC originally refered to: High Pressure Liquid...

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Transcript of High Performance Liquid Chromatography. HPLC originally refered to: High Pressure Liquid...

  • Slide 1
  • High Performance Liquid Chromatography
  • Slide 2
  • HPLC originally refered to: High Pressure Liquid Chromatography currently refers to: High Precision Liquid Chromatography high pressure to be able to use small particle size to allow proper separation at reasonable flow rates
  • Slide 3
  • Applications of Liquid Chromatography
  • Slide 4
  • Effect of Particle Size Effect of particle size of packing and flow rate upon height H in liquid chromatography. Column dimensions: 30cm x 2.4mm. Solute: N,N-diethyl-n- aminoazobenzene. Mobile phase: mixture of hexane, methylene chloride, isopropyl alcohol.
  • Slide 5
  • Slide 6
  • Effect of sample size on plate height
  • Slide 7
  • Components Solvent Reservoir and Degassing System Pumps Precolumns Sample Injection System Columns Temperature Control Detectors
  • Slide 8
  • Schematic of an apparatus for HPLC
  • Slide 9
  • Components Solvent Reservoir and Degassing System isocratic elution - single solvent separation teachnique gradiant elution - 2 or more solvents, varied during separation
  • Slide 10
  • Improvement in separation efficiency by gradient elution. Column: 1m x 2.1mm id, precision- bore stainless; packing: 1% Permaphase R ODS. Sample: 5 L of chlorinated benzenes in isopropanol. Detector: UV photometer (254 nm). Conditions: temperature 60 o C, pressure, 1200 psi.
  • Slide 11
  • Components Pumps 1000 - 6000 psi at rate of @ 3 mL/min reproducible to 2% screw driven syringe reciprocating pump
  • Slide 12
  • A reciprocating pump for HPLC.
  • Slide 13
  • Components Precolumns remove impurities from solvent saturates mobile phase with liquid of stationary pahse before the analytical column
  • Slide 14
  • Components Sample Injection System sample valve syringe
  • Slide 15
  • A sampling loop for liquid chromatography
  • Slide 16
  • Components Columns straight, 15 to 150 cm in length; 2 to 3 mm i.d. packing - silica gel, alumina, Celite
  • Slide 17
  • Components Temperature Control
  • Slide 18
  • Components Detectors single wavelength UV tuneable UV diode-array refractive index MS
  • Slide 19
  • Performances of LC Detectors
  • Slide 20
  • Ultraviolet detector cell for HPLC
  • Slide 21
  • Diode-Array Absorption Spectra Absorption spectra of the eluent from a mixture of three steroids taken at 5-second intervals.