Detection methods.

Abstract

Chromatography as a term was introduced by the Russian scientist Tswett, who separated plant pigments on a column containing calcium carbonate. It was based on the Greek word for color—“chromatos” and “graphein”—to write, together meaning “writing with color” because the separated pigments were seen on the column as zones of different colors. With time, the term “chromatography” became a description of separation of mixtures of different compounds. The current definition might be that “Chromatography is a separation method in which a mixture is applied as a narrow initial zone to a stationary, porous sorbent and the components are caused to undergo differential migration by the flow of the mobile phase, a liquid or a gas.” Most of the compounds separated in chromatographic processes in the early days of chromatography were colored compounds, which could be detected visually. This quickly became a limiting factor for the technique. Rapid development of chromatography created the need for more accurate detection techniques for a variety of analytes. Initially, this was achieved by chemical reactions of selected reagents with colorless analytes. The colored products of such reactions were detected and quantified spectrophotometrically using visible light, filters, and detecting devices connected to a recorder. A well-known example of such an approach is amino acid analysis first presented by Moore and Stein (). The separation and quantification was achieved by the use of ion-exchange chromatography and the detection was possible after the amino acids reacted with ninhydrin, yielding colored products. In the late 1960s and early 1970s, introduction of detection methods utilizing ultraviolet (UV) absorbance of many organic compounds permitted for a great expansion of analytical applications of chromatography.