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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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