HPLC,FPLC.pptVIP

HPLC and FPLC HPLC―High performance liquid chromatography. FPLC―Fast protein liquid chromatography. Chromatography Theory Even though each method utilizes different techniques to separate compounds,the principles are the same. Common to all: 1.a solid or a liquid supported on a solid as stationary phase. 2.A liquid or gas as mobile phase Chromatography Theory As the mobile phases passes through the stationary phase, it carries the components of the sample mixture with it. The components will not move at the same time because : 1:The components of the sample will be attached to the stationary phase, but there will also be a competing attraction for the mobile phase 2:Each components have its own characteristic balance of attraction to the mobile/stationary phase Column Chromatography HPLC HPLC―improved form of column chromatography The mobile phase is forced through the column under high pressure 500-6000psi HPLC have smaller packing material Smaller packing greater resistance to flow Need Higher pressures Normal Phase HPLC Stationary phase: polar, silica particles Mobile phase: non-polar solvent or mixture of solvents Polar compounds: Will have a higher affinity for the polar, stationary phase Will elute slower Non-polar compounds: Will have a higher affinity for the non-polar, mobile phase Will elute faster Reverse Phase HPLC Stationary phase: non-polar Non-polar organic groups are covalently attached to the silica stationary particles. Mobile phase: polar liquid or mixture of liquids Polar analytes will elute quicker than non-polar analytes Most common type of HPLC used today. The process of HPLC 1.Mobile phase pumped through column at high pressure. 2.Sample is injected into the system. 3.Separation occurs as the mobile phase and sample are pumped through the column. 4.Each sample component will elute from the column, one at a time, and will be detected. 5.The response of each component eluted will be displayed on a chart or computer screen.