Liquid Chromatography

Liquid Chromatography Liquid chromatographymass spectrometry (LC-MS, or alternatively HPLC-MS) is a chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry. LC-MS is a powerful technique used for many applications which has very high sensitivity and selectivity. Generally its application is oriented towards the general detection and potential identification of chemicals in the presence of other chemicals (in a complex mixture). Preparative LC-MS system can be used for fast and mass directed purification of natural products extracts and new molecular entities important to food, pharmaceutical, agrochemical and other industries. The limitations of LC-MS in urine analysis drug screening is that it often fails to distinguish between specific metabolites, in particular with hydrocodone and its metabolites. LC-MS urine analysis testing is used to detect specific categories of drugs. However, gas chromatography (GC-MS) should be used when detection of a specific drug and its metabolites is required.

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Principle The principle behind this technique is the differential adsorption of the various components of a mixture between two different phases that are as follows: Fixed or Stationary Phase The adsorbent is termed as the stationary phase. Suitable adsorbents that are commonly used are magnesium oxide, alumina, cellulose, paper, silica gel etc. Mobile or Moving Phase The liquid in which the substance is dissolved is termed as the mobile phase or eluent. The eluents employed are petroleum ether, carbon tetrachloride, benzene, alcohol etc. Their selection depends upon the relative solubilities of the components of the mixture in them. Column Chromatography This is the simplest chromatography based on the differential adsorption of the constituents of a mixture. A suitable adsorbent like alumina (Al2O3), taken in the form of a slurry in petroleum ether, constitutes the stationary phase. It is packed in a column prepared in a long burette-like glass tube with a stop-cock near the bottom. A plug of cotton or glass wool is placed at the bottom of the column to support the adsorbent. One-fourth of the tube is left empty. A loose plug of cotton or glass wool is then placed at the top of the adsorbent column. Process

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The mixture to be separated is taken in a suitable solvent and the solution is poured on the top of the column of the adsorbent. It is allowed to pass slowly through it and as it passes through the column the different constituents of the mixture get adsorbed to different extent, forming bands in different parts of the column. The components are then eluted out by a suitable solvent (known as eluent and which acts as a mobile phase). More than one eluent may be used in certain cases because the eluents dissolve different compounds selectively. The weakly adsorbed component will be eluted more rapidly than a more strongly adsorbed component. One may see separate bands in the column formed by different compounds of the mixture, if they are colored. Different components of the mixture are collected in the form of different fractions in separate conical flasks and distilled to get them in a pure form.

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