High Performance Liquid ChromatographyIntroductionHPLC systemFOUR TYPES OF LIQUID CHROMATOGRAPHYCOMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEMPicture of HPLC instrumentUses of HPLCHPLC Chromatograph injectorsHPLC columnsPicture of an HPLC columnWHAT AFFECTS SYSTEMSlide 12Several column types (can be classified as )Normal phaseReverse phaseSize exclusionIon exchangeSelectivity FactorTypes of Liquid Column Chromatography (LCC)Types of DetectorsEVALUATION PARAMETERSReferencesHigh Performance High Performance Liquid ChromatographyLiquid ChromatographyChem. 331Chem. 331IntroductionIntroduction HPLC is a form of liquid chromatography used to HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector.a pump, an injector, a separation column, and a detector.Compounds are separated by injecting a sample mixture Compounds are separated by injecting a sample mixture onto the column. The different component in the mixture onto the column. The different component in the mixture pass through the column at differentiates due to pass through the column at differentiates due to differences in their partition behavior between the mobile differences in their partition behavior between the mobile phase and the stationary phase. The mobile phase must phase and the stationary phase. The mobile phase must be degassed to eliminate the formation of air bubbles. be degassed to eliminate the formation of air bubbles.HPLC systemHPLC systemFOUR TYPES OF LIQUID FOUR TYPES OF LIQUID CHROMATOGRAPHYCHROMATOGRAPHYPartition chromatographyPartition chromatographyAdsorption, or liquid-solid Adsorption, or liquid-solid chromatographychromatographyIon exchange chromatographyIon exchange chromatographySize exclusion, or gel, chromatographySize exclusion, or gel, chromatographyCOMPOSITION OF A LIQUID COMPOSITION OF A LIQUID CHROMATOGRAPH SYSTEMCHROMATOGRAPH SYSTEMSolventSolvent Solvent Delivery System (Pump)Solvent Delivery System (Pump) InjectorInjector SampleSample ColumnColumn Detectors (Diode Array)Detectors (Diode Array) Waste CollectorWaste Collector Recorder (Data Collection)Recorder (Data Collection)Picture of HPLC instrumentPicture of HPLC instrumentUses of HPLC Uses of HPLC This technique is used for chemistry and biochemistry This technique is used for chemistry and biochemistry research analyzing complex mixtures, purifying chemical research analyzing complex mixtures, purifying chemical compounds, developing processes for synthesizing chemical compounds, developing processes for synthesizing chemical compounds, isolating natural products, or predicting physical compounds, isolating natural products, or predicting physical properties. It is also used in quality control to ensure the purity properties. It is also used in quality control to ensure the purity of raw materials, to control and improve process yields, to of raw materials, to control and improve process yields, to quantify assays of final products, or to evaluate product quantify assays of final products, or to evaluate product stability and monitor degradation. stability and monitor degradation. In addition, it is used for analyzing air and water pollutants, for In addition, it is used for analyzing air and water pollutants, for monitoring materials that may jeopardize occupational safety monitoring materials that may jeopardize occupational safety or health, and for monitoring pesticide levels in the or health, and for monitoring pesticide levels in the environment. Federal and state regulatory agencies use HPLC environment. Federal and state regulatory agencies use HPLC to survey food and drug products, for identifying confiscated to survey food and drug products, for identifying confiscated narcotics or to check for adherence to label claims.narcotics or to check for adherence to label claims.HPLC Chromatograph injectorsHPLC Chromatograph injectors The function of the injector is to place the sample into The function of the injector is to place the sample into the high-pressure flow in as narrow volume as possible the high-pressure flow in as narrow volume as possible so that the sample enters the column as a so that the sample enters the column as a homogeneous, low-volume plug. To minimize spreading homogeneous, low-volume plug. To minimize spreading of the injected volume during transport to the column, the of the injected volume during transport to the column, the shortest possible length of tubing should be used from shortest possible length of tubing should be used from the injector to the column. the injector to the column. When an injection is started, an air actuator rotates the When an injection is started, an air actuator rotates the valve: solvent goes directly to the column; and the valve: solvent goes directly to the column; and the injector needle is connected to the syringe. The air injector needle is connected to the syringe. The air pressure lifts the needle and the vial is moved into pressure lifts the needle and the vial is moved into position beneath the needle. Then, the needle is lowered position beneath the needle. Then, the needle is lowered to the vial. to the vial.HPLC columnsHPLC columnsThe column is one of the The column is one of the most important components most important components of the HPLC chromatograph of the HPLC chromatograph because the separation of because the separation of the sample components is the sample components is achieved when those achieved when those components pass through components pass through the column. The High the column. The High performance liquid performance liquid chromatography apparatus chromatography apparatus is made out of stainless is made out of stainless steel tubes with a diameter steel tubes with a diameter of 3 to 5mm and a length of 3 to 5mm and a length ranging from 10 to 30cm. ranging from 10 to 30cm. Normally, columns are filled Normally, columns are filled with silica gel because its with silica gel because its particle shape, surface particle shape, surface properties, and pore structure properties, and pore structure help to get a good separation. help to get a good separation. Silica is wetted by nearly every Silica is wetted by