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High.PerformanceUquid Chromatography

1.1 Introduction
"Chromatography is a method in which the components of a mixture are separated on an adsorbent column in a flowing system." 1 The adsorbent material, or stationary phase, first described by Tswett 1 in 1906, has taken many forms over the years, including paper, 2 thin layers of solids attached to glass plates, 3,4 immobilized liquids, 5 gels, 6 and solid particles packed in columns. 7 The flowing component of the system, or mobile phase, is either a liquid or a gas. Concurrent with development of the different adsorbent materials has been the development of methods more specific to particular classes of analytes. 6'8-a~ In general, however, the trend in development of chromatography
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Ion Exchange

Competition between liquid mobile phase and solid adsorbent

Competition between liquid mobile phase and 'liqui.d' stationary phase

Molecular sieving

'Lock and Key' mechanism

Competition between liquid mobile phase and ionic stationary phase

Figure 1.1

Classificationof chromatographicmodes according to the retention mechanism.

1.2 Classification of Liquid Chromatographic Methods


chromatography is not commonly used in modern HPLC. Instead, longchain (C18) "bonded-phase" columns have been developed in which the long alkyl chains are considered to behave like a liquid. Thus, the process is termed partition when the solute is transferred from the bulk of one phase into the bulk of the other, so that the solute molecules are completely surrounded by molecules of one phase. Under these conditions, the solutes are said to be in an isotropic environment. Ion-exchange chromatography (IEC) is based on the principle that opposites attract. Ion-exchange chromatography is used to separate charged analytes and therefore occurs as a result of interaction between a charged solute and an oppositely charged, solid stationary phase. Ion-exchange chromatography can be applied to any solute that can acquire a charge in solution. Thus, even carbohydrates, which are largely uncharged below pH 12, can be separated by ion-exchange chromatography at sufficiently high pH. Size-exclusion

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