621 chromatography

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62 1 CHROMATOGR APHY INTRODUCTION This chapter defines the terms and procedures used in chromatography and provides general information. Specific requ irements for chromatograp hic procedu res for drug substances and dosage for ms, including adsorbent and developing solvents, are given in the individual monographs. Chr omatograp hy is def ined as a procedu re by which solutes are separa ted by a dynamic diff eren tial migration process in a system consisting of two or more phases, one of which moves continuously in a given direction and in which t he indiv idual substances exhibit differ ent mobilities by reason of diff erences in adsorption, partition, solubility, vapor pressure, molecular size, or ionic charge density. The individual substances thus separated can be identified or determined by analytic al procedure s. The general chromatographic technique requires that a solute undergo distribution between two phases, one of them fixed (stationary phase), the other moving (mobile phase). It is the mobile phase that transfers the solute through the medium until it eventually emerges separated from other solutes that are eluted earlier or later. Generally, the solute is transported through the separation medium by means of a flowing stream of a liquid or a gaseous solvent known as the “eluant.” The stationary phase may act through adsorption, as in the case of adsorbents such as activated alumina and silica gel, or it may act by dissolving the solute, thus partitioning the latter between the stationary and mobile phases. In the latter process, a liquid coated onto an inert support, or chemically bonded onto silica gel, or directly onto the wall of a fused silica capillary, serves as the stationary phase. Partit ioning is t he pred ominant mechanism of separa tion in gas–liquid c hromatogr aphy, paper chromatogra phy, in forms of c olumn chromatograp hy, and in thin-layer c hromatogr aphy designated as liquid- liquid chromatograp hy. In practice, separations freq uently result fr om a combination of adsorption and partitioning effects . Ot her separa tion principles include ion exc hange , ion-p air for mation, s ize ex clusi on, hydropho bic interaction, and chiral recognition. The types of chromatography useful in qualitative and quantitative analysis that are employed in theUSP procedures are column, gas, paper, thin-layer, (including high-performance thin-layer chromatography), and pressurized liquid chromatography (commonly called high-pressure or high-performance liquid chromatography). Paper and thin-layer chromatography are ordinarily more useful for purposes of identification, because of their conv enience and simplicit y. Column chromatogr aphy off ers a wider choice of stationary phases and is us efu l for the s epara tion of indiv idual compounds, in quan tity , fr om mi xtures. Modern high -per for mance t hin-layer chromatography, gas chromatography, and pressurized liquid chromatography require more elaborate apparatus but usually provide high resolution and identify and quantitate very small amounts of material. Use of Reference Substances in Identity Tests — In paper an d thin-layer chromatograp hy, the ratio of the distance (this distance being measured to the point of maximum intensity of the spot or zone) traveled on the medium by a given compound to the distance traveled by the front of the mobile phase, from the point of application of the test substance, is designated as the R F value of the compound. The ratio between the distances traveled by a given compound and a reference substance is the R R value. R F values vary with the experimental conditions, and thus identification is best accomplished where an authentic specimen of the compound in question is used as a reference substance on the same chromatogram. For this purpose, chromatograms are prepared by applying on the thin-layer adsorbent or on the paper in a straight line, parallel to the edge of the chromatographic plate or paper, solutions of the substance to be identified, the authentic specimen, and a mixture of nearly equal amounts of the substance to be identified and the authentic specimen. Each sample application contains approximately the same quantity by weight of material to be chromatographed. If the substance to be identified and the authentic specimen are identical, all chromatograms agree in color and R F value and the mixed chromatogram yields a single spot; i.e., R R is 1.0. Chromatography Rf Silica gel flash grade 25-40&40-63 um,best result,best yields,ex-stock www.SorbeadIndia.com 24-09-2010 http://www.uspbpep.com/usp32/pub/… uspbpep.com//usp32nf27s0_c621.ht1/21

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