High PerformanceLiquid Chromatography

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HPLCoriginally refered to:High Pressure Liquid Chromatographycurrently refers to:High Precision Liquid Chromatography

high pressure to be able to use small particle size to allow proper separation at reasonable flow rates

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Applications of Liquid ChromatographyPartition chromatography

Adsorption, or liquid-solid

chromatography

Ion exchange chromatography

Size exclusion, or gel, chromatography

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ComponentsSolvent Reservoir and Degassing SystemPumpsPrecolumnsSample Injection SystemColumnsTemperature ControlDetectors

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Schematic of an apparatus for HPLC

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ComponentsSolvent Reservoir and Degassing Systemisocratic elution - single solvent separation techniquegradiant elution - 2 or more solvents, varied during separation

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Isocratic mixture of

A (aqueous buffer) and B (acetontrile)Isocratic elution: performed with a single solvent (or constant solvent mixture)

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Gradient ElutionGradient elution: continuous change of solventcomposition to increase eluent strength

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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.

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ComponentsPumps1000 - 6000 psi at rate of @ 3 mL/minreproducible to 2%screw driven syringereciprocating pump

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A reciprocating pump for HPLC.

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ComponentsPrecolumnsremove impurities from solventsaturates mobile phase with liquid of stationary phase before the analytical column

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ComponentsSample Injection Systemsample valvesyringe

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A sampling loop for liquid chromatography

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

http://www.youtube.com/watch?v=mPO7Tv2mIJUNormal phase Polar Stationary Phase and Nonpolar Solvent

Reverse phase Nonpolar Stationary Phase and Polar Solvent

Size exclusion (gel permeation) - Small molecules penetrate into the pores within the packing while larger molecules onlypartially penetrate the pores. The large molecules elute before the smaller molecules.

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Effect of Particle Size

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Columns Cont.

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ComponentsTemperature Control

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ComponentsDetectorssingle wavelength UVtuneable UVdiode-arrayrefractive indexMS

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Performances of LC Detectors

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Ultraviolet detector cell for HPLC

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Diode-ArrayAbsorption SpectraAbsorption spectra of the eluent from a mixture of three steroids taken at 5-second intervals.

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