HPLC.ppt
- 1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) • https://www.youtube.com/watch?v=eCj0cRtJvJg
- 2. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY • High Performance Liquid Chromatography (HPLC) is one of the most widely used techniques for identification, quantification and purification of mixtures of organic compounds. • In HPLC, as in all chromatographic methods, components of a mixture are partitioned between an adsorbent (the stationary phase) and a solvent (the mobile phase). • The stationary phase is made up of very small particles contained in a steel column. Due to the small particle size (3-5 um), pressure is required to force the mobile phase through the stationary phase.
- 3. • HPLC is really the automation of traditional liquid chromatography under conditions which provide for enhanced separations during shorter periods of time! • Probably the most widely practiced form of quantitative, analytical chromatography practiced today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!!
- 4. http://www.chemistry.nmsu.edu/Instrumentation/Waters_HPLC_MS_TitlePg.html HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- 6. • Stationary phases are particles which are usually about 1 to 20 m in average diameter (often irregularly shaped) – In Adsorption chromatography, there is no additional phase on the stationary phase particles (silica, alumina, Fluorosil). – In Partition chromatography, the stationary phase is coated on to (often bonded) a solid support (silica, alumina, divinylbenzene resin) Columns and Stationary Phases.
- 9. • Must do the following: – solvate the analyte molecules and the solvent they are in – be suitable for the analyte to transfer “back and forth” between during the separation process • Must be: – compatible with the instrument (pumps, seals, fittings, detector, etc) – compatible with the stationary phase – readily available (often use liters/day) – of adequate purity 99.5% solvents for HPLC • spectroscopic and trace-composition usually! – Not too compressible (causes pump/flow problems) • Free of gases (which cause compressability problems) The Mobile Phase in HPLC...
- 10. Optimization of Mobile Phase Polarity… B is organic solvent e.g. Acetonitrile Changing the mobile phase composition alters the separation.
- 13. • Usually 5 to 1000 L volumes, all directly onto the column – not much worry about capacity since the columns have a large volume (packed). – 1000 L 1.0 mL highest injection volume – Usually 20 t0 100 uL • Injector is the last component before the column(s) • Automatic injectors are available • Two positions, load and inject in the typical injector • Injection loop internal volume determines injection volume. Injection in HPLC
- 14. • Normal Phase: Separation of polar analytes by partitioning onto a polar, bonded stationary phase. • Reversed Phase: Separation of non-polar analytes by partitioning onto a non-polar, bonded stationary phase. • Adsorption: In Between Normal and Reversed. Separation of moderately polar analytes using adsorption onto a pure stationary phase (e.g. alumina or silica) • Ion Chromatography: Separation of organic and inorganic ions by their partitioning onto ionic stationary phases bonded to a solid support. • Size Exclusion Chromatography: Separation of large molecules based in the paths they take through a “maze” of tunnels in the stationary phase. Types of HPLC Separations
- 23. • Can use any UV-VIS with a special flow cell – Extra connections lead to band-broadening if UV-VIS is far from HPLC column exit. • Non-destructive, not-universal – not all compounds absorb light – can pass sample through several cells at several different wavelenghts • Usually zeroed at the start of each run using an electronic software command. You can have real-time zeroing with a reference cell. • 200-750 nm 1. Standard Absorbance Detector….
- 25. • The more common tool for research-grade HPLC instruments – quite versatile... • Advances in computer technology since ~1985 or so have lead to the development of Diode Array instruments • Non-destructive, non-universal • DAD scans a range of wavelengths every second or few seconds. At each point in the chromatogram one gets a complete UV-VIS spectrum! – Huge volumes of data – Detailed spectra for each peak and each region of each peak 2. Diode Array Detector (DAD)