For example, filtration takes advantage of substances being present in different states (solid vs. iquid); centrifugation relies on differences in density; and distillation makes use of differences in boiling points of the various components. Chromatography exploits differences in solubility and adsorption. The word chromatography, which is derived from two Greek words literally meaning color writing, was coined at the beginning of this century when the method was first used to separate colored components of plant leaves. Today, the name is a bit misleading, because most forms of chromatography do not depend on color.
Several types of chromatography are commonly used, among which are paper chromatography, thin-layer chromatography or TLC, liquid-liquid chromatography, gas chromatography, and high performance liquid chromatography or HPLC. Chromatography is so useful that some form can be found in most scientific laboratories around the world. For example, in forensic chemistry crime laboratories, the FBI maintains a library of chromatograms of inks that are used commercially. In the first case in which chromatography of inks were used, a man in Miami falsified travel and expense vouchers.
However, the ink pen he used had ink that wasnt available commercially until 3 years after the trips had taken place. The theory behind chromatography is to allow a mixture of different chemicals to be distributed or partitioned between a stationary phase and a mobile phase (eluent or solvent). The mobile phase may be a liquid or a gas; the stationary phase is typically a solid. As the mobile phase flows over the stationary phase, the components in the mixture are carried along. The more soluble a component is in the mobile phase the faster it will be transported along the stationary phase.
Adsorption refers to the ability of a substance to stick (or be adsorbed) to a surface. The more strongly a component is adsorbed to the stationary phase, the slower it will be transported by the mobile phase. As the mixture moves over the stationary phase, the components in the mixture move further and further apart into discrete zones. Paper chromatography uses ordinary filter paper (primarily cellulose) as the stationary phase. Thin-layer chromatography (abbreviated TLC) uses a thin glass plate coated with either aluminum oxide (alumina) or silica gel as the solid phase.
The mobile phase in both is a solvent chosen according to the properties of the components in the mixture. In paper chromatography, a drop of solution containing a substance or mixture of substances is spotted along a line near one end of a rectangular piece of filter paper. The paper is the stationary phase and the line is called the origin. The lower edge of the paper is placed in a developing solvent as the mobile phase. Capillary action causes the solvent to flow up the paper at a uniform rate creating a wet line across the paper.
This line is called the solvent front. When the solvent front reaches a spot, the components of the spot will begin to migrate upward with the mobile phase. Each component will have a characteristic chemical affinity for the paper and a characteristic chemical affinity for the solvent. These affinities are competitive: The components affinity for the paper tends to hold the component in one place, but its affinity for the solvent tends to make the component follow the solvent as it moves upward.
A component with a strong affinity for the paper and a weak affinity for the solvent will move more slowly than a component with a weaker affinity for the paper and a stronger affinity for the solvent. TLC works in similar manner. The affinity of a substance for the stationary and mobile phases is characteristic of that substance. Different substances will have different competitive affinities. Since each component of a mixture will have its own characteristic affinities, each component will travel up the paper at its own characteristic rate.
If the paper is sufficiently large, all the components can be separated by the time the solvent front has reached the top of the paper and each component will appear as a separate spot. The chromatographic paper will now contain a vertical array of colored spots arranged according to their characteristic rates of ascent. It is possible to describe the position of spots (so the substances that have separated) in terms of their retention factor, the Rf value (Figure 1).