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”

Effect of Particle Size “Effect of particle size of packing and flow rate upon height H in liquid chromatography. Column dimensions: 30cm x 2.4mm. Solute: N,N-diethyl-n- aminoazobenzene. Mobile phase: mixture of hexane, methylene chloride, isopropyl alcohol.”

“Effect of sample size on plate height”

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 teachnique –gradiant elution - 2 or more solvents, varied during separation

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 pahse 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

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.”