HPLC.pptx
- 1. Chromatography • Chromatography is a technique to separate mixtures of substances into their components on the basis of their molecular structure and molecular composition. • This involves a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). • The mobile phase flows through the stationary phase and carries the components of the mixture with it. • Sample components that display stronger interactions with the stationary phase will move more slowly through the column than components with weaker interactions. • This difference in rates cause the separation of variuos components. • Chromatographic separations can be carried out using a variety of stationary phases, including immobilized silica on glass plates (thin-layer chromatography), volatile gases (gas chromatography), paper (paper chromatography) and liquids (liquid chromatography).
- 2. HPLC – High Performance Liquid Chromatography (Principle, type, instrumentation and applications) • High performance liquid chromatography (HPLC) is basically a highly improved form of column liquid chromatography. • Instead of a solvent being allowed to drip through a column under gravity, it is forced through under high pressures of up to 400 atmospheres. That makes it much faster. • All chromatographic separations, including HPLC operate under the same basic principle; separation of a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation.
- 3. Type of HPLC • Normal Phase HPLC: This method separates analytes on the basis of polarity. NP-HPLC uses polar stationary phase and non-polar mobile phase. Therefore, the stationary phase is usually silica and typical mobile phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of these. Polar samples are thus retained on the polar surface of the column packing longer than less polar materials. • Reverse Phase HPLC: The stationary phase is nonpolar (hydrophobic) in nature, while the mobile phase is a polar liquid, such as mixtures of water and methanol or acetonitrile. It works on the principle of hydrophobic interactions hence the more nonpolar the material is, the longer it will be retained.
- 4. • Size-exclusion HPLC: The column is filled with material having precisely controlled pore sizes, and the particles are separated according to its their molecular size. Larger molecules are rapidly washed through the column; smaller molecules penetrate inside the porous of the packing particles and elute later. • Ion-Exchange HPLC: The stationary phase has an ionically charged surface of opposite charge to the sample ions. This technique is used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are used to control elution time.
- 5. Instrumentation As shown in the schematic diagram in Figure above, HPLC instrumentation includes a pump, injector, column, detector and integrator or acquisition and display system. The heart of the system is the column where separation occurs.
- 7. 1. Solvent Resorvoir : Mobile phase contents are contained in a glass reservoir. The mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample. 2. Pump : A pump aspirates the mobile phase from the solvent reservoir and forces it through the system’s column and detector. Depending on a number of factors including column dimensions, particle size of the stationary phase, the flow rate and composition of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be generated.
- 8. 3. Sample Injector : The injector can be a single injection or an automated injection system. An injector for an HPLC system should provide injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi). 4. Columns : Columns are usually made of polished stainless steel, are between 50 and 300 mm long and have an internal diameter of between 2 and 5 mm. They are commonly filled with a stationary phase with a particle size of 3–10 µm. Columns with internal diameters of less than 2 mm are often referred to as microbore columns. Ideally the temperature of the mobile phase and the column should be kept constant during an analysis.
- 13. 5. Detector : The HPLC detector, located at the end of the column detect the analytes as they elute from the chromatographic column. Commonly used detectors are UV-spectroscopy, fluorescence, mass- spectrometric and electrochemical detectors. 6. Data Collection Devices : Signals from the detector may be collected on chart recorders or electronic integrators that vary in complexity and in their ability to process, store and reprocess chromatographic data. The computer integrates the response of the detector to each component and places it into a chromatograph that is easy to read and interpret.
- 16. Interpretation
- 17. • Retention: In an elution chromatographic separation substances differ from each other only in their residence time in or at the stationary phase. From this the following time definitions arise: • The total retention time ( or ) is the time, which is needed by a sample component to migrate from column inlet (sample injection) to the column end (detector). • The dead time t0 is the time required by an inert compound to migrate from column inlet to column end without any retardation by the stationary phase. Consequently, the dead time is identical with the residence time of the sample compound in the mobile phase.
- 18. • The net retention time ( or ) is the difference between total retention time and dead time. That is the time the sample component remains in the stationary phase. • The capacity factor k’ is a measure of the position of a sample peak in the chromatogram. It is specific for a given substance. k’ depends on the stationary phase, the mobile phase, the temperature, quality of the packing etc. • The relative retention α, also known as separation factor, is the ratio between two capacity factors, where the figure in the denominator is the reference compound. The relative retention describes the ability of a system of stationary and mobile phase to discriminate between two compounds. It is independent of column construction (length, quality of packing) and flow velocity. It depends on the temperature and the properties of the mobile and stationary phases. Impurities in the mobile phase (e.g. water content) strongly influence the relative retention.
- 19. Applications • Pharmaceutical Applications 1. To control drug stability. 2. Tablet dissolution study of pharmaceutical dosages form. 3. Pharmaceutical quality control. • Environmental Applications 1. Detection of phenolic compounds in drinking water. 2. Bio-monitoring of pollutants.
- 20. • Applications in Forensics 1. Quantification of drugs in biological samples. 2. Identification of steroids in blood, urine etc. 3. Forensic analysis of textile dyes. 4. Determination of cocaine and other drugs of abuse in blood, urine etc. • Food and Flavour 1. Measurement of Quality of soft drinks and water. 2. Sugar analysis in fruit juices. 3. Analysis of polycyclic compounds in vegetables. 4. Preservative analysis. • Applications in Clinical Tests 1. Urine analysis, antibiotics analysis in blood. 2. Analysis of bilirubin, biliverdin in hepatic disorders. 3. Detection of endogenous Neuropeptides in extracellular fluid of brain etc.