HPLC/High Performance Liquid Chromatography/High Pressure Liquid Chromatography/Working&Instrumental скачать в хорошем качестве

HPLC/High Performance Liquid Chromatography/High Pressure Liquid Chromatography/Working&Instrumental

This video describes the principle of HPLC. The various types of mechanism that could be used with HPLC is listed. It is used to separate various components present in the mixture by the difference in the affinity of each component with the stationary phase and mobile phase. HPLC video explains the various types of columns and different types of chromatographic mechanism it can follow for each column. The instrumentation and working of HPLC, The different types of columns used are explained. The different types of detectors used in HPLC is discussed in detail. The advantages and disadvantages of this method is used. The various applications are explained. High performance liquid chromatography is a technique used to separate the components/compounds present in a mixture based On the distribution of the components between the stationary phase and the mobile phase. It is used to separate the liquid samples and solid samples dissolved in suitable solvents. It is not suitable for gaseous samples. All mechanisms of chromatography can be adopted by choosing an appropriate column Adsorption chromatography Normal phase chromatography Reverse phase chromatography Size exclusion chromatography (SEC) / Gel permeation chromatography (GPC) Ion-exchange chromatography Affinity chromatography Mobile phase in HPLC Solvent or eluent reservoirs One reservoir – isocratic elution Two or more reservoirs – gradient elution moves from the reservoir to the mixing chamber regulated by the solvent proportionality valve along with injected sample, the mobile phase is passed through the stationary phase at a constant flow rate/constant pressure. Mixing chamber mixes the solvents coming from the reservoirs Mobile phase delivery system in HPLC solvent reservoirs solvent degassing unit micro-filter microprocessor - mobile phase mixing chamber controlled high pressure pump High pressure pump in HPLC Isocratic elution – One reservoir and one pump Mixture of two solvents – binary system Mixture of three solvents – ternary system Mixture of four solvents – quaternary system Gradient elution – Two or more reservoirs and independent pump for each reservoir Single pump in conjunction with solvent proportionality valve Sample injection port/valve injectori in HPLC – sample is injected using microsyringe into the injection port as a sharp pulse Stationary phase in HPLC Stationary phase is packed in the column uniformly at a very high density – increases resistance to flow of mobile phase – requires high pressure pump It results in small plate height – large number of theoretical plates per unit length – gives rise to high separation efficiency Adsorption chromatography and Normal phase chromatography - small rigid uniform particles of silica or alumina Reverse phase chromatography – surface modified silica particles Size exclusion chromatography/ Gel permeation chromatography – rigid porous polymeric beads (polystyrene) Ion-exchange chromatography – ion-exchange resins based on synthetic or natural polymers Affinity chromatography – consists of specific ligands coupled or immobilised on beads of agarose or other suitable solvents Column in HPLC Analytical Column – stainless steel or glass column containing packed bed of stationary phase– 25 cm long & 3-4mm diameter Guard column/pre-column – placed between the sample injector and analytical column – to protect the main column from damage. Detectors in HPLC Solvent property detector – detects the change in the property of the mobile phase such as refractive index or conductance - refractive index detector or conductance meter Solute property detector – detects the property of the solute – UV/Visible detector, Photodiode array detector, fluorescence detector or electrochemical detector Refractive index detector (RID) – used for SEC/GPC/samples without UV/visible absorption No deflection of the light beam when the pure mobile phase passes through both the reference compartment and sample compartment. Deflection of the light beam proportional to the amount of the solute component, when the pure mobile phase passes through the reference compartment and mobile phase mixed with solute through the sample compartment. Conductance meter detector for HPLC– used for ion-exchange chromatography UV/Visible/Photodiode array detector for HPLC (absorbance detectors) – samples with UV/Visible absorption Data processor – It processes the signals coming the detector and sends to the recorder. Recorder – Records the chromatogram by plotting the time against the detector signal response. Advantages of HPLC High speed compared to column and TLC– within 10 to 30 minutes.Accurate and highly reproducible.Quantitative sample recovery.Fully automated operation.High sensitivity Disadvantages of HPLC No universal detector.Less separation efficiency Applications of HPLC