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Published byTabitha Barrett Modified over 9 years ago
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High Performance Liquid Chromatography
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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
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“Applications of Liquid Chromatography” Partition chromatography Adsorption, or liquid-solid chromatography Ion exchange chromatography Size exclusion, or gel, chromatography
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Components Solvent Reservoir and Degassing System Pumps Precolumns Sample Injection System Columns Temperature Control Detectors
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“Schematic of an apparatus for HPLC”
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Components Solvent Reservoir and Degassing System –isocratic elution - single solvent separation technique –gradiant 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 Elution Gradient elution: “continuous change of solvent composition 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% Permaphase R ODS. Sample: 5 L of chlorinated benzenes in isopropanol. Detector: UV photometer (254 nm). Conditions: temperature 60 o C, pressure, 1200 psi.
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Components Pumps –1000 - 6000 psi at rate of @ 3 mL/min –reproducible to 2% –screw driven syringe –reciprocating pump
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“A reciprocating pump for HPLC.”
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Components Precolumns –remove impurities from solvent –saturates mobile phase with liquid of stationary phase before the analytical column
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Components Sample Injection System –sample valve –syringe
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“A sampling loop for liquid chromatography”
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Components Columns –straight, 15 to 150 cm in length; 2 to 3 mm i.d. –packing - silica gel, alumina, Celite http://www.youtube.com/watch?v=mPO7Tv2mIJU Normal 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 only partially 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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Components Temperature Control
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Components Detectors –single wavelength UV –tuneable UV –diode-array –refractive index –MS
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Performances of LC Detectors
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Ultraviolet detector cell for HPLC
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Diode-Array Absorption Spectra “Absorption spectra of the eluent from a mixture of three steroids taken at 5-second intervals.”
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