Chromatography | Research & Encyclopedia Articles

Chromatography

Chromatography is a technique used in analytical chemistry to separate and identify components of mixtures. The name comes from the Greek term for "color writing" because this method was originally used to separate colored samples. Today a variety of chromatography methods work by separating the individual components of mixtures so that each one can be analyzed and identified. Instruments used to perform chromatography are known as chromatographs, which have become essential pieces of equipment in chemical laboratories. With these devices, scientists can tell what chemical compounds are present in complex mixtures such as smog, cigarette smoke, petroleum products, or even coffee aroma.

The first chromatograph was invented just after the turn of the twentieth century by Russian botanist Mikhail Semenovich Tsvett (1872-1919), whose name (sometimes rendered as "Tswett") coincidentally means "color" in Russian. Tsvett was born in Italy of a Russian father and Italian mother. After studying in Switzerland and doing some research in St. Petersburg, Tsvett settled in Warsaw, which at that time belonged to the Russian empire. During his years in Poland, Tsvett's botanical research led to his great moment of insight. Tsvett was looking for a method of separating a mixture of plant pigments, which are chemically very similar to each other. To isolate different types of chlorophyll, Tsvett trickled a mixture of dissolved pigments through a glass tube packed with calcium carbonate powder. As the solution washed downward, each pigment stuck to the powder with a different degree of strength, creating a series of colored bands. Each band of color represented a different substance, and Tsvett referred to the colored bands as a chromatogram. He also suggested that the technique (now called adsorption chromatography) could be used to separate colorless substances. Although Tsvett published a report of this work in the early 1900s, chemists paid very little attention to it. For one thing, the report was written in Russian, which few Western chemists of the time read. Also, the technique of chromatography may have seemed too simplistic to chemists, who were used to relying on lengthy extraction, crystallization, or distillation to separate mixtures. But within a few years, Tsvett's technique was rediscovered by German organic chemist Richard Martin Willstatter (1872-1942), who was also studying chlorophyll. By introducing chromatography to Western European scientists, Willstatter helped establish one of the most versatile analytical techniques known to chemistry.

Willstatter went on to define the major types of chlorophyll and discover the importance of magnesium in the chlorophyll molecule. Soon chromatography was found to work on almost all kinds of mixtures, including colorless ones, as Tsvett had predicted. Absorbing powders were discovered that perform better than calcium carbonate for separating ordinary molecules. Also, compounds known as zeolites were introduced to separate individual ions, or electrically charged particles, in a process called ion-exchange chromatography. American chemist Frank Harold Spedding adapted this technique to the separation of rare-earth metals. In the 1930s, synthetic resins were developed for complex ion-exchange processes. During World War II, life rafts were equipped with survival kits that contained resins for removing most salts from seawater.

The most dramatic advance in the history of chromatography took place in 1944, when scientists discovered that a strip of porous filter paper could substitute for the column of absorbing powder. In this technique, called paper chromatography, a drop of the mixture to be separated is placed on the paper, then one edge is dipped into a solvent. The solvent spreads across the paper, carrying the mixture's components with it. When the components are finished spreading, the paper is dried and sprayed with a reagent that reveals a change in color. Because the components move at different speeds, they show up as distinct, physically separated spots that can be cut out with scissors and further analyzed. The paper method is a type of partition chromatography, which is based on differences in solubility rather than differences in adsorption. One of its advantages is that it requires only a small sample of material. Paper chromatography was invented by two British biochemists, Archer John Porter Martin (1910-) and Richard Laurence Millington Synge (1914-). Martin was the son of a physician, while Synge's father was a stockbroker. Both scientists studied at Cambridge University, where Martin earned his Ph.D. in 1936 and Synge in 1941. Martin began working in the university's nutritional laboratory, where he investigated problems related to vitamin E. For a few years, he also became involved in a study of the felting of wool. In 1941, Martin and Synge began working together on proteins, which are made up of chains of amino acids. Martin and Synge were trying to characterize a particular protein by determining the precise numbers of each amino acid present. Amino acids are so similar to each other, however, that the problem of separating them had defeated a whole generation of biochemists. Martin and Synge's development of paper chromatography to solve this problem was an instant success, not only on amino acids but also on various other mixtures. The two scientists were awarded the Nobel Prize in chemistry in 1952 for their work. Martin and Synge's research led to a number of other important scientific advances. After Synge determined the structure of an antibiotic peptide called Gramicidin-S, Frederick Sanger (1918-) used paper chromatography to figure out the structure of the insulin molecule--not only the number of particular amino acids in it, but also the order in which they occurred. Insulin is now used to control blood sugar levels in people afflicted with diabetes.

The same technique was used by Melvin Calvin (1911- 1997) during the 1950s to discover the complex series of reactions that enable green plants to convert solar energy into the chemical energy stored in food. Working with green algal cells, Calvin interrupted the photosynthetic process at different stages by plunging the cells into alcohol. Then he crushed them and separated their components via paper chromatography. Calvin was thus able to identify at least ten different intermediate products that had been created within a few seconds. Paper chromatography was also used by Austrian-American biochemist Erwin Chargaff (1905-), who modified the technique to study the components of the nucleic acid molecule. His research revealed four components, or nitrogenous bases, that occur in pairs. British biochemists Watson and Crick later used these results to work out the structure of DNA (deoxyribonucleic acid). The genetic material of humans, other animals, and plants is made of DNA, which is passed on from generation to generation and is responsible for all inherited traits.

In addition to inventing paper chromatography, Archer Martin developed another technique called gas chromatography, which enables chemists to separate mixtures of gases, or substances that can be vaporized or gasified by heat. Instead of a liquid solvent, helium gas is usually used to force the mixture through a column and separate the gaseous components. Martin and his colleague A. T. James first used gas chromatography to microanalyze fatty acids. The widespread acceptance of gas chromatography is unique in the laboratory instrumentation field. Today it is used in almost every branch of the chemical industry, particularly in the production of petrochemicals from oil and natural gas. One of the most common fixtures in biochemical laboratories is "GCMS" analytical equipment, which uses gas chromatography to separate individual components from complex organic mixtures, then uses mass spectrometry to identify each component.

Recently, chromatography has evolved into even more sophisticated analytical techniques. In thin-layer chromatography, for example, an alumina gel, silica gel, or other finely divided solid is spread onto a glass plate in a thin, uniform layer that takes the place of filter paper in the chromatographic process. This technique is not only faster than paper chromatography, but it can also separate smaller quantities of pure components. It is often used in the pharmaceutical industry to isolate penicillin and other antibiotics. A number of new products for chromatography were featured at the 1998 Pittsburgh Conference and Exposition on Analystical Chemistry and Applied Spectroscopy. For example, the Dionex company showed its latest ion exchange technology, the EG40, which can separate charged molecules. This device promises to be a valuable tool in the analysis of water quality and environmental contaminants and on-line monitoring of raw materials and production processes.