Chromatographic techniques separate mixtures by exploiting differences between the physicochemical properties of their components.
Techniques such as gas chromatography can provide detailed quantitative and qualitative analyses using samples that are only a few microliters in volume. These tests can detect traces of pesticide residues in food and drugs in blood and urine samples. In industry, chromatography is used to check the purity of products and to control pollution by testing the contents of effluents before they are released to the environment.
Scientists in laboratories use columns packed with silica to separate the substances in product mixtures from experimental syntheses; a thin layer of silica on a glass plate can be used to the same effect for minute quantities of products.
Chromatographic methods were developed as a means of purifying complex natural products that were difficult or impossible to extract by other methods. As early as the 19th century, dye chemists tested the contents of dye vats by dipping the edges of rags into the liquid and watching the individual dyes form separate bands as the liquid seeped through the rag. In 1903, the Russian botanist Mikhail Tsvet used a column packed with calcium carbonate powder to split mixtures of plant pigments into colored bands. Tsvet called his technique chromatography, from the Greek words meaning "color writing."
Principles of chromatography
In any form of chromatographic technique, the components of a mixture are split between a mobile carrier phase, which can be a gas or a liquid, and a stationary phase, which can be liquid or solid. The mobile phase flows through or along the stationary phase, but the two do not mix.
The various chemical compounds in the mixture pass back and forth between the mobile phase and the stationary phase as the one flows past the other. Components of the mixture that have a stronger affinity for the stationary phase than for the mobile phase spend more of their time trapped by the stationary phase, so they pass through more slowly than components that have a greater affinity for the mobile phase.
The time taken for a component to pass though the stationary phase is called its retention time; it depends on the nature of the mobile and stationary phases, the length of the path through the stationary phase, and the flow rate of the mobile phase. Often, the retention time is stated relative to that of a calibrating substance.
Column chromatography
Two British chemists, Archer Martin and Richard Synge, developed column chromatography in the 1940s. Their goal was to separate the components of mixtures of amino acids derived from wool. At first, they tried to effect this separation by making two immiscible solvents flow in opposite directions while in contact with each other. The amino acids separated according to differences in their relative affinities for the two solvents.
Martin and Synge had limited success with their efforts until they hit on the idea of binding one of the solvents—water—to finely powdered silica (SiO2) packed in a vertical glass column. While their equipment resembled that used by Tsvet some 40 years before, the technique differed in that the stationary phase was a liquid. The mobile phase was trichloromethane (chloroform, CHCl3), which does not mix with water.
A chromatography column is prepared by pouring a suspension of hydrated silica in solvent into a cylindrical glass column that has a tap at the bottom and a sintered-glass disk just above. The disk allows solvent to pass through while preventing silica from escaping through the tap. When the silica has settled as a layer, solvent is drained from the column until the upper surface of the silica is just wet with solvent. The sample solution is then added, and solvent is drained until the sample soaks into the top of the silica bed. The operator then adds more solvent at the top of the column and partially opens the tap to control its rate of flow through the column. Those substances that have a high affinity for the solvent of the mobile phase move down the column more rapidly than other components. The liquid that emerges from the column is collected in small portions that yield the individual components of the mixture when the solvent is removed by evaporation.
