Clinical Chemistry 5 Chromatography and Electrophoresis

Quantitative assessment of the relative

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Unformatted text preview: peak positions with those of standards. Quantitative assessment of the relative concentrations of components is obtained from peak area comparisons. Column performance is indicated by comparison with standards. Chromatographic Techniques Chromatographic Liquid chromatography techniques – Classical methods used columns packed with Classical any stationary phase any – Fractions of the eluate are caught and Fractions analyzed analyzed Thin Layer chromatography Thin TLC is a variant of column chromatography A thin layer of sorbent (such as alumina, silica thin gel, or cellulose) is uniformly coated on a glass or plastic plate or Each sample to be analyzed is applied as a spot Each near the edge of the plate near The mobile phase (solvent) is usually placed in a The closed container until the atmosphere is saturated with solvent vapor saturated One edge of the plate is placed in the solvent One The solvent migrates up the thin layer by The capillary action → dissolving and carrying sample molecules molecules Sample components are identified by comparison Sample with standards on the same plate with The distance of a component migrates compared with The the distance the solvent front moves is called the ratio of fronts → Rf of Rf = distance leading edge of the component moves distance distance solvent front moves distance Each sample component Rf is compared with the Rf of standards of High Performance Liquid Chromatography Chromatography HPLC Modern liquid chromatography uses Modern pressure for fast separations pressure A pump forces the mobile phase through pump the column at a much greater velocity that do gravity flow columns do Detectors: – Visible, UV, fluorescence, refractive index, Visible, electrochemical electrochemical Gas Chromatography Gas Gas chromatography is used to separate mixtures of Gas compounds that are volatile or can be made volatile compounds The mobile phase is a gas – samples are partitioned The between gaseous mobile phase and a liquid stationary phase phase The sample is vaporized upon injection and is separated The in relationship to the substances boiling point in Compounds with the higher boiling point will spend more Compounds time in the stationary phase. time The effluent passes through a detector that produces an The electrical signal proportional to concentration...
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This document was uploaded on 03/22/2014.

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