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% Permaphase R ODS. Sample: 5  L of chlorinated benzenes in isopropanol. Detector: UV photometer (254 nm). Conditions: temperature 60 o C, pressure, 1200 psi.

Components Pumps – psi at rate 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 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.”