- High-Performance Liquid Chromatography
- HPLC Separation Modes
- Gas Chromatography
- Ion Chromatography
- Thin-layer Chromatography
Chromatography is a technique that is widely used for the separation of gases, liquids, or dissolved substances. After being separated, the individual constituents can be identified or purified through standard techniques. Its accuracy and ease of use make chromatography a fundamental procedure in analytic chemistry.
The originator of chromatography as an analytic technique was the Russian botanist Mikhail Tsvet, whose name is sometimes transliterated as Tswett (1872-1920). In 1906, while working on plant pigments, Tsvet filled a tube with calcium carbonate (CaCO3). He poured a mixture of pigments dissolved in petroleum ether into the tube and then poured a solvent through the tube. As they trickled through the tube, the various pigments adhered to the calcium carbonate. Each pigment had a different degree of attraction to the calcium carbonate, so the weakly attached pigments were displaced by the ones that were more strongly attracted. These pigments were forced downwards until they too adhered to the calcium carbonate. This process caused the different pigments to separate at different levels within the tube, resulting in a series of colored bands. For this reason, Tsvet called the process chromatography, which means “written in color” in Greek.
Through the use of this method, Tsvet was able to determine that extract of leaves contained two chlorophylls, four xanthophylls, and carotene. Unfortunately, the utility of chromatography was not widely appreciated for many years thereafter. Although Tsvet published a number of papers in German, his major work on the subject was written in Russian, and chromatography received little notice in the scientific world until it was used in 1931 by Richard Kuhn (1900 – 1967) and his coworkers to resolve several pigments, including carrot carotene and egg yolk xanthophyll, into their isomeric components. Also of considerable importance was Richard Willstätter’s (1872 – 1942) use of chromatography for research on chlorophyll.
As more chemists turned to the use of chromatography, aluminum oxides and silica gel replaced calcium carbonate. Current practice generally entails collecting successive fractions of the liquid after they have passed through the column rather than separating fractions within the tube. Chromatography is also used for separating ions of different compounds through a process called ion exchange. This technique also has a practical application: The calcium and magnesium ions responsible for making water “hard” can be removed by running the water through a column filled with zeolite (hydrated aluminosilicate). The calcium and magnesium atoms are adsorbed by the zeolite, and their place is taken by sodium ions originally present in the zeolite.
Analytical chemistry became easier and more accurate through the development of paper chromatography. The basic idea underlying paper chromatography is quite old; the Roman writer Pliny the Elder (23 – 79 CE) noticed that different liquid substances separated when they were absorbed by a sheet of paper. In the mid-19th century, Friedrich Runge (1795 – 1867) wrote two books that described how a solution of mixed colored compounds formed concentric rings when it was dripped onto filter paper. In the 1940s, two British chemists, Archer John Porter Martin (1910 ) and Richard Laurence Millington Synge (1914 – 1994) developed paper chromatography as a means of separating the various amino acids that were the basic constituents of proteins. To do this, Martin and Synge put a small drop of amino acid mixture near one end of a strip of filter paper and allowed it to dry. The end was then placed in a solvent, and the solvent moved towards the dried area by capillary action. The solvent then carried the various amino acids up the paper at different rates according to their solubility, causing them to separate in a linear fashion. Each amino acid could then be identified by matching it with the position of a known amino acid that had undergone the same treatment. In the 1950s, paper chromatography was used to good effect for the study of photosynthesis as it occurs in green plants.
The last major phase in the development of chromatography occurred in 1953 when Martin and his coworkers invented gas chromatography. A typical apparatus for gas chromatography consists of a very long tube containing a solid substance like kieselguhr (a fine filtering medium based on diatomaceous earth) or a nonvolatile liquid. A mixture of gases (often vaporized liquids) is pushed through the column by a carrier gas such as hydrogen. This causes the various gases to pass through the tube at different speeds because they are adsorbed at different rates by the solid or liquid in the tube. The passage of the gases at different speeds allows them to be identified (through the use of a flame detector or measurements of thermal conductivity) as they exit in sequence from the tube.