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High Performance Liquid Chromatography

Learn about the applications and effects of particle size in High Performance Liquid Chromatography. Explore the components and techniques used to improve separation efficiency.

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High Performance Liquid Chromatography

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  1. High Performance Liquid Chromatography

  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

  3. “Applications of Liquid Chromatography”

  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.”

  5. “Effect of sample size on plate height”

  6. Components Solvent Reservoir and Degassing System Pumps Precolumns Sample Injection System Columns Temperature Control Detectors

  7. “Schematic of an apparatus for HPLC”

  8. Components Solvent Reservoir and Degassing System • isocratic elution - single solvent separation teachnique • gradiant elution - 2 or more solvents, varied during separation

  9. Improvement in separation efficiency by gradient elution. Column: 1m x 2.1mm id, precision-bore stainless; packing: 1% PermaphaseR ODS. Sample: 5mL of chlorinated benzenes in isopropanol. Detector: UV photometer (254 nm). Conditions: temperature 60oC, pressure, 1200 psi.

  10. Components Pumps • 1000 - 6000 psi at rate of @ 3 mL/min • reproducible to 2% • screw driven syringe • reciprocating pump

  11. “A reciprocating pump for HPLC.”

  12. Components Precolumns • remove impurities from solvent • saturates mobile phase with liquid of stationary pahse before the analytical column

  13. Components Sample Injection System • sample valve • syringe

  14. “A sampling loop for liquid chromatography”

  15. Components Columns • straight, 15 to 150 cm in length; 2 to 3 mm i.d. • packing - silica gel, alumina, Celite

  16. Components Temperature Control

  17. Components Detectors • single wavelength UV • tuneable UV • diode-array • refractive index • MS

  18. Performances of LC Detectors

  19. Ultraviolet detector cell for HPLC

  20. Diode-ArrayAbsorption Spectra “Absorption spectra of the eluent from a mixture of three steroids taken at 5-second intervals.”

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