High Performance Liquid Chromatography HPLC originally refered to:

High Performance Liquid Chromatography HPLC  originally refered to:

High Performance Liquid Chromatography 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 Applications of Liquid

Chromatography Partition chromatography Adsorption, or liquid-solid chromatography

Ion exchange chromatography Size exclusion, or gel, chromatography Components

Solvent Reservoir and Degassing System Pumps Precolumns Sample Injection System

Columns Temperature Control Detectors Schematic

of an apparatus for HPLC

Components Solvent Reservoir and Degassing System isocratic elution - single solvent separation technique

gradiant elution - 2 or more solvents, varied during separation Isocratic mixture of A (aqueous buffer)

and B (acetontrile) Isocratic elution: performed with a single solvent (or

constant solvent mixture) Gradient Elution

Gradient elution: continuous change of solvent composition to increase eluent

strength Improvement in separation efficiency by gradient elution. Column: 1m x

2.1mm id, precision-bore stainless; packing: 1% PermaphaseR ODS. Sample: 5L of chlorinated benzenes in isopropanol.

Detector: UV photometer (254 nm). Conditions: temperature 60oC, pressure, 1200 psi.

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

reciprocating pump A reciprocating pump for HPLC.

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

Components Sample Injection System sample valve syringe

A sampling loop for liquid chromatography Components http://www.youtube.com/watch?v=mPO7Tv2mIJU

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

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. Effect of Particle Size Columns Cont.

Components Temperature Control Components

Detectors single wavelength UV tuneable UV diode-array refractive index

MS Performances of LC Detectors Ultraviolet

detector cell for HPLC Diode-Array Absorption

Spectra Absorption spectra of the eluent from a mixture of

three steroids taken at 5-second intervals.

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