Date: July 29, 2014
High performance liquid Chromatography is an
analytical technique method by which the compounds are physically separated prior to measurement the main purpose of chromatography is to separate and quantify the target sample in the matrix. Chromatogram is a plot of detector signal output versus time, and Mobile phase is a liquid that analyte or mixture of liquids moves the solute through the column, Stationary phase packing material of the column, which is the immobile phase involved in the chromatographic process. Peak is the visual representation on the chromatogram based on the detector’s electrical response due to the presence of a sample component inside the flow cell. Retention time is the time taken by the analyte peak to reach the detector after sample injection. Qualitation is analysis process which is designed to identify the components of a substance or mixture, quantization is an analysis process which is designed to determine the amounts or proportion of the components of a substance. The three forms of High performance liquid chromatography most often used are based on mechanism of partition, adsorption and ion exchange. The separation of ionic solutes takes place on the basis of ion exchange on stationary phases with charged functional groups. The functional groups typically are quaternary ammonium groups for anion exchange and negatively charged groups like sulphonates for cation exchange. The corresponding counter ions are located in Vicinity of the functional groups and can be exchanged with other ions of the same charge in the mobile phase. Thus, various ionic components of the sample can be separated based on their differential affinities towards the immobilized stationary and the
liquid mobile phase.
A pumping system, an injector, a chromatographic column with or without a column temperature controller, a detector and a data acquisition system (a computer, an integrator or a chart (recorder) are the essential components of the equipment. For ion exchange chromatography a suppressor column is installed between main column and detector. The mobile phase is supplied from one or several reservoirs and flows through the column, usually at a constant rate, and then through the detector.
Any part of the system that is in contact with the mobile phase should be constructed of materials inert to corrosive components of the mobile phase. The entire system dead volume has to be kept at the minimum. The tubing length and diameter of plumbing between the injector, column and detector has to be kept at the minimum. Higher volumes in these connections lead to increased dispersion and tailing of peaks.
The pumping systems deliver metered amounts of the mobile phase from the solvent reservoirs to the column through high pressure tubing and fittings. Modern systems consist of one or more computer-controlled metering pumps that can be programmed to vary the ratio of mobile phase components, as is required for gradient elution chromatography, or to make an Isocratic mobile phase (i.e., mobile phases having a fixed ratio of solvents). The system should be capable of delivering the mobile phase at a constant rate with minimal fluctuations over extended periods of time. Pumps may be provided with a mechanism for ‘bleeding’ the system of any entrapped air.
The samples that are to be chromatographed are injected, either manually. are injected, either manually. loop injectors, or automaticity by auto samplers. An auto sampler consists of a carousel or rack to hold sample vials with tops that have a pierceable septum or stopper and an injection device to transfer samples from the vials to a loop from which it is loaded in to the chromatograph. Some auto samplers can be programmed to control sample volume, the number of injections and loop rinse cycles, the interval between injections, and other operating variables like temperature. Manual partial filling of loops is not recommended because of poorer injection volume precision.
STATIONARY PHASES AND COLUMNS
chromatography are silica, alumina or porous graphite, are used as stationary phase in normal phase chromatography, wherein separation is based on the principle of adsorption and/or mass distribution, chemically modified silica, polymers or porous graphite, are used as stationary phase in reversed-phase chromatography, where in separation is based principally on partition of the molecules between the mobile phase and the stationary phase,For most pharmaceutical analysis separation is achieved by partitioning of compounds in the test solution between the mobile and stationary phases. Systems consisting of polar stationary phases and non-polar mobile phases are described as normal phases while the opposite arrangement (i.e., polar mobile phases and non polar stationary phases) are called reversed-phases.
In reversed-phase chromatography utilizing chemically modified silica, the silanol groups of silica on the surface of the support react with various silane reagents to produce covalently bound silyl derivatives covering a varying number of active sites on the surface of the support. The nature of the bonded phase influences the separation properties of the chromatographic system. Commonly used bonded phases are octyl (Cs), octadecyl (CIS), phenyl (C6Hs), cyanopropyl (CN), aminopropyl (N’B:2) and diol.
The temperature of the mobile phase and the column must be kept constant during an analysis. Most separations are performed at ambient conditions, but columns may be heated to give higher efficiency; In order to reduce the possibility of degradation of the stationary phase or of occurrence of changes in the composition of the mobile phase, heating of the columns above 60° is not recommended. In some cases a particular commercial brand of column that has been found suitable is mentioned, but such statements do not imply that a different but equivalent commercial brand cannot be used. It may be necessary with a particular chromatograph to modify the conditions specified in the monograph but it should been sured that comparable results are obtained under the modified conditions.
UV/VISIBLE spectrophotometers,including diode array detectors, are the most commonly employed detectors. Abeam of UV radiation passes through the flow cell and into the detector. As compounds elute from the column, they pass through the cell and absorb the radiation, resulting in measurable energy level changes. The wavelength setting is specified in the individual monograph. Fluorescence spectrophotometers, differential refractometers, electrochemical detectors, mass spectrometers, light scattering detectors, radioactivity detectors or other special
detectors may also be used. Fluorimetric detectors are sensitive to compounds that are fluorescent or that can be converted to fluorescent derivatives. Differential refractometer detectors measure the difference between the refractive index of the mobile phase alone and that of the mobile phase containing Chromatographer compound as it emerges from the column. Electrochemical detectors are suitable for measuring nanogram quantities of easily oxidisable compounds like phenols and Catechols. Conductivity detector is the detector of choice in ion exchange chromatography. UV-visible detectors for absorbing species, indirect UV-visible detectors, amperometric detectors and fluorescence detectors are also employed for specific applications.
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