Post on 18-Dec-2015
High PerformanceLiquid
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
PumpsPrecolumnsSample Injection SystemColumnsTemperature ControlDetectors
“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% 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 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-ArrayAbsorption Spectra“Absorption spectra of the eluent from a mixture of three steroids taken at 5-second intervals.”