What is HPLC? High Performance Liquid Chromatography High Pressure Liquid Chromatography (usually...
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What is HPLC? High Performance Liquid Chromatography High Pressure Liquid Chromatography (usually true] Hewlett Packard Liquid Chromatography (a joke) High Priced Liquid Chromatography (no joke) 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 today due to the wide range of molecule types and sizes which can be separated using HPLC or variants of HPLC!! Slide 2 This highly efficient technique uses solvent under pressure to elute the column. Although generally used as an analytical system, larger capacity columns having good resolving power can be used in preparative systems. The general arrangement and operation bears more resemblance to gas chromatography, but HPLC is the more powerful analytical technique as it permits analysis of non-volatile and thermally labile compounds. However, development of the technique had to await the technology for preparing the specialized stationary phases which is so crucial for success. Slide 3 Slide 4 Liquid Column Chromatography A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid. With the proper solvents, packing conditions, some components in the sample will travel the column more slowly than others resulting in the desired separation. Slide 5 Basic liquid chromatography modes are named according to the mechanism involved: 1.Liquid/Solid Chromatography (adsorption chromatography) A.Normal Phase LSC B.Reverse Phase LSC 2.Liquid/Liquid Chromatography (partition chromatography) A.Normal Phase LLC B.Reverse Phase LLC 3.Ion Exchange Chromatography 4.Gel Permeation Chromatography (exclusion chromatography) Four Basic Liquid Chromatography Slide 6 Importance of HPLC in Pharmaceutical Analysis Drug manufacturing control requires high level and intensive analytical and chemical support of all stages to ensure the drug's quality and safety. The pharmacopeia constitutes a collection of recommended procedures for analysis and specifications for the determination of pharmaceutical substances, excipients, and dosage forms that is intended to serve as source material for reference or adaptation by anyone wishing to fulfill pharmaceutical requirements. The most important analytical technique used during the various steps of drug development and manufacturing is the separation technique: High Performance Liquid Chromatography (HPLC). Slide 7 Mechanism of separation HPLC is a form of liquid chromatography used to separate compounds that are dissolved in solution. The different components in the mixture pass through the column at different rates due to differences in their adsorption (LSC) or partition (LLC) behavior between the mobile phase and the stationary phase. Slide 8 Composition of the System of HPLC Solvent reservoirs Solvent Delivery System (Pump) Injector for introducing sample Column Detectors Waste Collector Recorder (Data Collection) Slide 9 Slide 10 Slide 11 The solvents used for HPLC must be highly pure, contain no solid matter and will require degassing immediately before use by sonification or by displacement of dissolved gases with helium. Any formation of air bubbles within the system totally upsets the pressure maintenance. Criteria of solvents for HPLC use Slide 12 DEGASSING: The practice of removing air from the mobile phase is called degassing which can be achieved by bubbling He (Helium) gas into the mobile phase and this degassing removes dissolved oxygen from the Mobile Phase. The presence of oxygen in mobile phase causes bubble formation resulting in air in the flow system and pump pressure will change causing spike in the chromatogram (due to air bubble formation in the detector cell). Slide 13 The Equipment of HPLC HPLC Column: The column is a stainless steel tube packed with a special stationary phase consisting of extremely fine, regularly sized, spherical particles with a very high ratio of surface area to mass, usually in the range 200-300 m 2 g -1. Owing to the small size of the particles, typically 5-10m in diameter, any solvent contained in the matrix of each particle is close to the external solvent and equilibration can take place readily. This particular feature makes HPLC much more efficient than column chromatography using supports, in which the much larger particles have deep interstices predominantly filled with stagnant, non-equilibrating solvent. Slide 14 Typically, analytical columns are 10-25 cm long with an internal diameter of 4-6 mm and may be surrounded by a thermo stated bath for extremely accurate work. Owing to the small size of the column the `dead space' in the connections between the outlet and the detector must be kept to an absolute minimum in order to avoid remixing of components after elution from the column. The columns are designed to be reusable, and so the set-up always possesses a pre-column filter to remove particulate or polymeric matter before it contaminates the main column. Slide 15 Picture of an HPLC column Slide 16 Several column types based on the stationary phases Normal phase column Reverse phase column Size exclusion column Ion exchange column Chiral column Slide 17 Normal phase In this column type, the retention is governed by the interaction of the polar parts of the stationary phase and solute. For retention to occur in normal phase, the packing material (stationary phase) must be more polar than the mobile phase with respect to the sample. Example of stationary phase: silica and Alumina Slide 18 Normal Phase Stationary Phase - POLAR Mobile Phase -NON POLAR + - + - + - + - + - + - + - + - + - + - + - + Slide 19 Reverse phase In this column the packing material is relatively nonpolar and the solvent is polar with respect to the sample. Retention is the result of the interaction of the nonpolar components of the solutes and the nonpolar stationary phase. Typical stationary phases are nonpolar hydrocarbons, waxy liquids, or bonded hydrocarbons (such as C18, C8, etc.) and the solvents are polar aqueous-organic mixtures such as methanol-water or acetonitrile-water. Slide 20 Reverse Phase Stationary Phase - NON POLAR Mobile Phase - POLAR - + - - + + Slide 21 In reverse phase chromatography, a highly polar solvent system is used for elution. Under such conditions polar compounds prefer to stay in the mobile phase and are eluted before non-polar materials which have a greater affinity for the stationary phase-the reverse of usual adsorption systems (normal phase chromatography). Slide 22 Size exclusion In size exclusion the HPLC column is consisted of substances which have controlled pore sizes and is able to be filtered in an ordinary phase according to its molecular size. Small molecules penetrate into the pores within the packing while larger molecules only partially penetrate the pores. The large molecules elute before the smaller molecules. Slide 23 Ion exchange In this column type the sample components are separated based upon attractive ionic forces between molecules carrying charged groups of opposite charge to those charges on the stationary phase. Separations are made between a polar mobile liquid, usually water containing salts or small amounts of alcohols, and a stationary phase containing either acidic or basic fixed sites. Slide 24 Chiral column Another very exciting development of the column uses stationary phases in which the surface has been covalently linked to a chiral material. Such chiral bonded phase supports permit the analytical separation of enantiomers, and the potential for such chiral columns seems boundless. These stationary phases are known as customized stationary phases which are really expensive. Slide 25 HPLC Pumps As a consequence of the highly packed nature of the stationary phase in HPLC columns, pressures of anything up to 7000 psi are necessary to force the eluting solvent along the column which is effectively performed in HPLC by using pump. The pump must be capable of maintaining a constant flow of solvent with no pressure surges and it must also be constructed of material which can withstand a wide range of organic solvents. The pump is also designed to permit the precise choice of a particular rate from a wide range of flow rates. Slide 26 Elution may be with a single solvent or a mixture of solvents mixed at a certain ratio, when the process is described as isochratic elution, or the composition of the eluting system can be varied with time to permit gradient elution. The mixing of solvents for gradient elution may be carried out either at the low pressure side of the system, before the pump, or in the high pressure part. Both arrangements require microprocessor control. Elution Process Slide 27 Introduction of sample to the column Samples for analysis are introduced onto the column by injection through a multiport valve which permits precise, repeatable sample loading with minimal disturbance of solvent flow. Basically, a solution of the sample is injected into an isolated, fixed volume loop of tubing which, on turning the valve, becomes part of the solvent delivery system. Sample in excess of that required to fill the loop is led to waste so-called external loop injection ports permit variation of the volume of sample introduced into the column. Slide 28 Valve-Type Injection in HPLC Widely used in HPLC. Allows reproducible introduction of sample into the pressurized mobile phase without interruption of flow even at high temperature. Inject valve has two position: (a) Load (Fill) (b) Inject Valve in the load position. Before injecting the sample the valve is turned to the load position. 23 4 1 56 3 1 2 4 5 6 Slide 29 Automated Injection Sample is introduced from a vial held in a sample carousel using a syringe assembly. Valves are automatically actuated used