Chromatography: Difference between revisions
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Latest revision as of 16:01, 28 July 2024
Chromatography is a chemical purification process that involves separating substances that have different affinities for other substances in their environment. These substances can be solids or gases as well as the solvent containing the substances. Solutes with different distribution coefficients (the ratio of their concentration in one substance to their concentration in another) will be differentially associated with the substances in their environment and can thus be gradually separated. The solutes do not "collect" in a particular solvent, solid or gas, but rather come to a state of reversible, dynamic equilibrium in which their molecules are constantly moving between their solvent and other phases, to a degree determined by the distribution coefficient. In chromatographic purification, one of the phases in the solutes' environment is mobile (typically liquid) and another is stationary.
Liquid-solid chromatography
In liquid-solid chromatography, the stationary phase is a solid surface, able to adsorb solute molecules is substituted for one solvent. The distribution coefficient then becomes an adsorption coefficient,
- KA = (amount of solute A adsorbed per unit surface area) / (concentration of solute A in solution)
Solutes differ in their adsorption to the solid surface (both from each other and from their solvent) primarily because of differences in polarity, which in turn result from electronegativity differences within the molecules[1].
Column chromatography
One example of liquid-solid chromatography is column chromatography, in which solutes with different affinity constants (KAs) move at different speeds through a vertical column filled with solids like glass or sand (the stationary phase). The solution (liquid phase) is repeatedly poured from the top of the column, and bands of solute develop as the solutes are cyclically adsorbed and redissolved. The bands move downward and separate from each other, with the less tightly adsorbed solutes (having large KAs) moving slower than low KA substances. The solutes can be collected separately (elution) as they reach the end of the column[1].
Thin layer chromatography
Another method of liquid-solid chromatography is thin layer chromatography, which can only handle small volumes of solute and is more useful for simple identification of solution components. The solid is a film on a sheet of glass or plastic; the solution is pressed lightly onto the plate. When the plate is placed into a layer of solvent, which then moves upwards, carrying the solutes, by capillary action. The solutes will move less than the solvent, and they can be distinguished and identified by a constant retention factor Rf, the ratio of the distance traveled by the solute to that traveled by the particular solvent used[1]