High Performance Liquid Chromatography HPLC originally refered to
- Slides: 31
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: 1 m x 2. 1 mm id, precision-bore stainless; packing: 1% Permaphase. R ODS. Sample: 5 m. L of chlorinated benzenes in isopropanol. Detector: UV photometer (254 nm). Conditions: temperature 60 o. C, pressure, 1200 psi.
Components Pumps – 1000 - 6000 psi at rate of @ 3 m. L/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=m. PO 7 Tv 2 m. IJU 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. ”
- High performance liquid chromatography hplc machine
- Principle of hplc ppt
- High performance liquid chromatography introduction
- Isocratic and gradient elution in hplc
- Principle of hplc
- Chromatography what is
- Fid detector principle
- Gas liquid chromatography
- Types of liquid chromatography
- Void volume
- Commercial liquid dielectrics
- Liquid liquid extraction diagram
- Band broadening and column efficiency
- Priming hplc
- Rührapparatur
- Hplc werking
- Hplc detector types
- Clarity hplc software
- "for internal use only"
- M tswett pronunciation
- Kelebihan dan kekurangan hplc
- Hplc cihazı kısımları
- Principle of hplc
- Hplc detector types
- Hplc lecture
- Factors affecting column chromatography
- Chromato
- Stationary and mobile phase
- Hplc detector types
- Hplc instrumentation diagram
- Block diagram of hplc
- Problemas comunes en hplc