Chromatography

Chromatography is the single most valuable technique available for separating and purifying very SMALL quantities of a mixture -- exactly the situation where crystallisation or distillation would struggle. The name itself (Greek chroma, colour, and graphein, to write) reflects its origin: it was first developed in 1906 by M.S. Tswett, a Russian botanist, who separated the different coloured pigments making up chlorophyll by passing a petroleum-ether extract of leaves through a narrow glass tube tightly packed with calcium carbonate -- the different pigments visibly separated into distinct coloured bands (or zones) as they travelled down the tube at different rates. The technique's usefulness was very quickly recognised to extend well beyond coloured substances, and it is now applied equally to colourless mixtures.

The underlying principle in every case is the same: chromatography relies on the SELECTIVE DISTRIBUTION of the components of a mixture between two phases -- a stationary phase, which can be either a solid or a liquid, and a moving (mobile) phase, which can be either a liquid or a gas. When the stationary phase is a SOLID, separation happens on the basis of adsorption (different compounds sticking to the solid surface to different degrees); when the stationary phase is a LIQUID, separation happens on the basis of partition (different compounds distributing themselves differently between the two immiscible liquid phases). This lets chromatography be defined, generally, as a technique for separating a mixture by the differential movement of its individual components through a porous medium, driven by a moving solvent. Five specific methods fall under this one umbrella: column chromatography (CC), thin-layer chromatography (TLC), paper chromatography (PC), gas-liquid chromatography (GLC), and ion-exchange chromatography.

Adsorption chromatography -- the shared principle behind both CC and TLC -- relies on different compounds in a mixture being adsorbed onto a solid adsorbent to different DEGREES; silica gel and alumina are the two adsorbents most commonly used, and because different components of the mixture are held with different strength, they end up travelling different DISTANCES over the stationary phase as the mobile phase moves through it. In column chromatography, specifically, this happens inside a long glass column fitted with a stopcock near its lower end: a plug of cotton or glass wool is placed at the bottom to hold the packing in place, and the column is then uniformly packed with a suitable adsorbent (activated aluminium oxide/alumina, magnesium oxide and starch are all used besides silica gel), forming the stationary phase. The mixture to be separated is loaded on top of this packed column, and an eluent (a liquid, or a mixture of liquids) is then allowed to flow slowly down through the column; because different components are adsorbed to different degrees, they separate as they travel -- the most strongly adsorbed components stay retained near the top of the column, while more weakly adsorbed components travel further down before they, too, become held, and complete separation results.

Thin-layer chromatography is a second adsorption-based technique, distinguished mainly by letting even very minute quantities of a mixture be separated cleanly. A sheet of glass (called a chromoplate, or TLC plate) is coated with a thin, uniform layer of an adsorbent -- cellulose, silica gel or alumina. Once that coating has dried, a small drop of the mixture to be tested is placed near one edge of the plate, which is then stood upright inside a closed jar containing the eluent (solvent). The eluent is drawn steadily up through the thin adsorbent layer by capillary action, and as it rises it carries the different components of the mixture up along with it, to different distances, depending on each component's own degree of adsorption onto the plate's coating. How far any one spot has travelled, relative to how far the solvent front itself travelled, is expressed as that spot's retention factor: Rf = (distance moved by the substance from the baseline) / (distance moved by the solvent from the baseline). Spots belonging to coloured compounds are simply visible directly on the developed plate; spots belonging to colourless compounds instead have to be located either by viewing the plate under UV light, by exposing it to iodine vapour/crystals, or by spraying it with an appropriate chemical reagent that reacts to reveal the spots. …