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HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) When particles of small diameter (microns) are used as a stationery phase support, the technique is called HPLC. H * Column effeciency is inversely proportional to column-packing particle size. **Better resolution **High performance P LC High pressure to pump liquids through an efficient column. It is the most widely used form of LC in assaying many analytes: -amino acids, -peptides and proteins, -carbohydrates, -lipids, -nucleic acids, -vitamins, -hormones, -drugs eg. antibiotics, antiepileptics, antidepressants. Solvent Reservoir Solvents used in the mobile phase are contained in solvent reservoirs. The reservoirs may be glass bottles or flasks into which “feed lines” to the pump are inserted. Solvent Clarity The solvent must be free of particulate matter as impurities can affect column pump Detection system The Pumping System It is used to aspirate the mobile phase from the solvent reservoir and forcing it through the column. The main features of a good pumping system are: Capable of producing high pressure outputs No pulses (cyclic variations in pressure) The dual piston reciprocating pump It is a pump that delivers a fixed volume of solvent (0.01-20ml) onto the column by a piston driven by a motor. The piston is moved by a motorized crank (cam) which is assymetrical, and the entry of the solvent to the column is regulated by check valves. The dual piston reciprocating pump How is the HPLC pump operated Modes of solvent application Isocratic mode: #The mobile phase remains constant throughout the run. # used for separation of compounds with similar structures. # The isocratic mobile phase can be a single solvent (eg. methanol) or a mixture of solvents delivered from a single reservoir. Gradient mode # The mobile phase composition is changed during the run in either a stepwise or a continuous fashion. # The gradient profile can be generated by different techniques. The Injector An aliquot of the sample (0.5- 50µL) is introduced into the column via an injector. The most widely used type is the fixed loop injector. It consists of a stainless-steel loop of a fixed volume, that can be filled with the sample. It has a valve switching system that creates two positions: a- a fill or loading position ,and, b- An inject position. The Fixed Loop Injector**see animation2 In the fill (loading position), the sample is introduced with a syringe into the external loop of the injector. In the inject mode, the sample loop is rotated into the flowing stream of the mobile phase. This results in the flushing of the sample into the column. ----------------------------------------------N.B. Auto samplers are also available. They are extremely precise and can be programmed for automated operation. Columns Most analytical HPLC columns in clinical laboratories are fabricated from stainless steel. The following items will be referred to: * Guard columns. * Column dimensions. * Column packing. After a number of separations, a guard column is routinely replaced. Column Dimensions The internal diameters (ID) of the columns range from 0.3-5mm. The length of the columns range from 50-250mm. Column end fittings (with zero dead volumes) and frits to retain the support particles are used to connect the column to the injector on the inlet and the detector on the outlet end. In addition, capillary columns (of 0.1-0.5mm ID) and 10-50cm in length are available. They are fabricated through coating the inner wall of a fused- silica tube with a thin film of a liquid phase. Column Packing Columns are packed with small diameter (3-10µm) particles made of an inert material. The surfaces of these particles are covered by the stationery phase. As packing size is decreased, efficiency and pressure requirements are increased. Analytical columns are packed with a variety of stationery phases, providing enormous versatility in the separation processes. The most popular is the bonded phase packing. Here the stationery phase is bonded chemically to the surface of silica particles through silica ester linkage. The columns packed in this way can act by various separation mechanisms, depending on the type of the stationery phase bonded to the particles. The C18 type stationery phase in reversed phase partition chromatography is commercially called octadecyl silane (ODS). In normal phase partition chromatography the mobile phase is relatively non-polar eg.hexane. In reversed phase partition chromatography the mobile phase is relatively polar eg.water\methanol. N.B. Sephadex, sepharose and other non-rigid gels are of limited use in HPLC as they cannot withstand very high pressures. Detectors Many detectors have been developed in HPLC. The most common are: Photometric (Visible\UV) detectors. Fluorometric detectors. Electrochemical detectors. Refractory index detectors. # A key component in these detectors is the flow cell through which the elute from the chromatographic column passes. Spectrophotometric (Visible\UV) detectors These measure the radiant energy absorbed by analytes as they elute from the chromatographic column. Most organic compounds absorb in the UV region (190-400nm) of the spectrum. Some compounds absorb in the visible region of the spectrum (400700). The magnitude of light absorption and the wavelength at which absorption of light occurs are a function of the molecular structure and the concentration of the eluted compounds. Fluorometric Detectors These detect analytes that emit light (fluorescent compounds). Fluorescence occurs when a molecule absorbs light at one wavelength and becomes excited; then when it returns to its non- excited (ground) state, it emits light of another wavelength specific for that analyte. The analyte detected by fluorometers must have a fluorescent character or treated so that it becomes fluorescent (i.e. derivatized). Fluorometric Detectors Derivatization can be done before the sample enters the column OR to the eluent eluted from the column. Thus either a pre-column or post- column reactor, chemically tags a compound for subsequent detection. Example: tagging amino acids with fluorescamine tag. Fluorometric Detectors Light sources in fluorometers: Deuterium lamps Xenon arc lamps Laser lamps Electrochemical detectors Here an electro-active analyte enters the flow cell where it is oxidized or reduced at an electrode surface under a constant potential. It is useful for measuring catecholamines. Derivatization by putting electrochemically active tags (eg. bromine) for compounds as unsaturated fatty acids and prostaglandins can be done. Computer System Control. Data Processing. Data Processing. Analyte identification The retention time, or volume, at which an unknown solute elutes from the column is matched to that of a reference (standard compound). Analyte Quantification A calibration (standard) curve must be used for measuring analyte concentration. External calibration Internal calibration External calibration Reference (standard) solutions containing known quantities of same analyte to be measured are processed in a manner identical to the samples containing the analyte. A calibration curve of peak height or (area) versus concentration is constructed and used to calculate the concentration of the analyte in the samples. Internal calibration Also called internal standardization. A different compound called internal standard, is added to each reference solution and each sample. By plotting the ratio of the peak height or (area) of the calibrator to the peak height or (area) of internal standard versus the concentration of the calibrator, a calibration curve that corrects for systematic losses is constructed. The curve is then used to compute the concentration of the analyte in unknown samples. Sample preparation Sample concentration/purification: This is done using extraction cartridges (1-3ml propylene syringes that contain the stationery phase). Sample derivatization: eg. a- Treating amino acids with ninhydrin to produce a color for photometric detection. b- Tagging analytes with bromine for electrochemical detection. c- Tagging analytes with fluorescamine tag for fluorometeric detection.