History of chromatography
Encyclopedia
The history of chromatography spans from the mid-19th century to the 21st. Chromatography
, literally "color writing", was used—and named— in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll
(which is green) and carotenoid
s (which are orange and yellow). New forms of chromatography developed in the 1930s and 1940s made the technique useful for a wide range of separation process
es and chemical analysis tasks, especially in biochemistry
.
through an inert material to create separation of the solution components based on differential adsorption
—is sometimes attributed to German chemist Friedlieb Ferdinand Runge, who in 1855 described the use of paper to analyze dye
s. Runge dropped spots of different inorganic chemicals onto circles of filter paper already impregnated with another chemical, and reactions between the different chemicals created unique color patterns. According to historical analysis of L. S. Ettre, however, Runge's work had "nothing to do with chromatography" (and instead should be considered a precursor of chemical spot tests such as the Schiff test
).
In the 1860s, Christian Friedrich Schönbein
and his student Friedrich Goppelsroeder published the first attempts to study the different rates at which different substances move through filter paper. Schönbein, who thought capillary action
(rather than adsorption) was responsible for the movement, called the technique capillary analysis, and Goppelsroeder spent much of his career using capillary analysis to test the movement rates of a wide variety of substances. Unlike modern paper chromatography, capillary analysis used reservoirs of the substance being analyzed, creating overlapping zones of the solution components rather than separate points or bands.
Work on capillary analysis continued, but without much technical development, well into the 20th century. The first significant advances over Goppelsroeder's methods came with the work of Raphael E. Liesegang: in 1927, he placed filter strips in closed containers with atmospheres saturated by solvents, and in 1943 he began using discrete spots of sample adsorbed to filter paper, dipped in pure solvent to achieve separation. This method, essentially identical to modern paper chromatography, was published just before the independent—and far more influential—work of Archer Martin and his collaborators that inaugurated the widespread use of paper chromatography.
. Tsvet applied his observations with filter paper extraction to the new methods of column fractionation
that had been developed in the 1890s for separating the components of petroleum
. He used a liquid-adsorption column containing calcium carbonate
to separate yellow, orange, and green plant pigment
s (what are known today as xanthophylls, carotenes, and chlorophyll
s, respectively). The method was described on December 30, 1901 at the 11th Congress of Naturalists and Doctors (XI съезд естествоиспытателей и врачей) in Saint Petersburg
. The first printed description was in 1903, in the Proceedings of the Warsaw
Society of Naturalists, section of biology. He first used the term chromatography in print in 1906 in his two papers about chlorophyll in the German botanical journal, Berichte der Deutschen Botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for the German Botanical Society. Interestingly, Mikhail's surname "Цвет" means "color" in Russian, so there is the possibility that his naming the procedure chromatography (literally "color writing") was a way that he could make sure that he, a commoner in Tsarist Russia, could be immortalized.
In a 1903 lecture (published in 1905), Tsvet also described using filter paper to approximate the properties of living plant fibers in his experiments on plant pigments—a precursor to paper chromatography
. He found that he could extract some pigments (such as orange carotene
s and yellow xanthophyll
s) from leaves with non-polar solvents, but others (such as chlorophyll
) required polar solvents. He reasoned that chlorophyll was held to the plant tissue by adsorption
, and that stronger solvents were necessary to overcome the adsorption. To test this, he applied dissolved pigments to filter paper, allowed the solvent to evaporate, then applied different solvents to see which could extract the pigments from the filter paper. He found the same pattern as from leaf extractions: carotene could be extracted from filter paper using non-polar solvents, but chlorophyll required polar solvents.
Tsvet's work saw little use until the 1930s.
and Richard Laurence Millington Synge. By "the marrying of two techniques, that of chromatography and that of countercurrent solvent extraction", Martin and Synge developed partition chromatography to separate chemicals with only slight differences in partition coefficient
s between two liquid solvents. Martin, who had previously been working in vitamin chemistry (including attempts to purify vitamin E
), began collaborating with Synge in 1938, bring his experience with equipment design to Synge's project of separating amino acids. After unsuccessful experiments with complex countercurrent extraction machines and liquid-liquid chromatography methods where the liquids move in opposite directions, Martin hit on the idea of using silica gel in columns to hold water stationary while an organic solvent flows through the column. Martin and Synge demonstrated the potential of the methods by separating amino acids marked in the column by the addition of methyl red
. In a series of publications beginning in 1941, they described increasingly powerful methods of separating amino acids and other organic chemicals.
In pursuit of better and easier methods of identifying the amino acid constituents of peptides, Martin and Synge turned to other chromatography media as well. A short abstract in 1943 followed by a detailed article in 1944 described the use of filter paper as the stationary phase for performing chromatography on amino acids: paper chromatography
. By 1947, Martin, Synge and their collaborators had applied this method (along with Fred Sanger's reagent
for identifying N-terminal residues) to determine the pentapeptide sequence of Gramicidin S
. These and related paper chromatography methods were also foundational to Fred Sanger's effort to determine the amino acid sequence of insulin
.
(shared with Synge, for their earlier chromatography work) Martin announced the successful separation of a wide variety of natural compounds by gas chromatography. (German chemist Fritz Prior had achieved limited success with gas chromatography, separating oxygen
and carbon dioxide
, in 1947 during his Ph.D. research. The method of Martin and James, however, became the basis for subsequent developments in gas chromatography.)
The ease and efficiency of gas chromatography for separating organic chemicals spurred the rapid adoption of the method, as well as the rapid development of new detection methods for analyzing the output. The thermal conductivity detector
, described in 1954 by N. H. Ray, was the foundation for several other methods: the flame ionization detector
was described by J. Harley, W. Nel, and V. Pretorius in 1958, and James Lovelock
introduced the electron capture detector
that year as well. Others introduced mass spectrometers to gas chromatography in the late 1950s.
The work of Martin and Synge also set the stage for high performance liquid chromatography
, suggesting that small sorbent particles and pressure could produce fast liquid chromatography techniques. This became widely practical by the late 1960s (and the method was used to separate amino acids as early as 1960).
occurred in the 1940s, and techniques advanced rapidly in the 1950s after the introduction of relatively large plates and relatively stable materials for sorbent
layers.
and Meir Wilchek
were awarded the Wolf Prize in Medicine
for the invention and development of affinity chromatography
and its applications to biomedical sciences.
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
, literally "color writing", was used—and named— in the first decade of the 20th century, primarily for the separation of plant pigments such as chlorophyll
Chlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
(which is green) and carotenoid
Carotenoid
Carotenoids are tetraterpenoid organic pigments that are naturally occurring in the chloroplasts and chromoplasts of plants and some other photosynthetic organisms like algae, some bacteria, and some types of fungus. Carotenoids can be synthesized fats and other basic organic metabolic building...
s (which are orange and yellow). New forms of chromatography developed in the 1930s and 1940s made the technique useful for a wide range of separation process
Separation process
In chemistry and chemical engineering, a separation process, or simply a separation, is any mass transfer process used to convert a mixture of substances into two or more distinct product mixtures, at least one of which is enriched in one or more of the mixture's constituents. In some cases, a...
es and chemical analysis tasks, especially in biochemistry
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
.
Precursors
The earliest use of chromatography—passing a mixed solutionSolution
In chemistry, a solution is a homogeneous mixture composed of only one phase. In such a mixture, a solute is dissolved in another substance, known as a solvent. The solvent does the dissolving.- Types of solutions :...
through an inert material to create separation of the solution components based on differential adsorption
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
—is sometimes attributed to German chemist Friedlieb Ferdinand Runge, who in 1855 described the use of paper to analyze dye
Dye
A dye is a colored substance that has an affinity to the substrate to which it is being applied. The dye is generally applied in an aqueous solution, and requires a mordant to improve the fastness of the dye on the fiber....
s. Runge dropped spots of different inorganic chemicals onto circles of filter paper already impregnated with another chemical, and reactions between the different chemicals created unique color patterns. According to historical analysis of L. S. Ettre, however, Runge's work had "nothing to do with chromatography" (and instead should be considered a precursor of chemical spot tests such as the Schiff test
Schiff test
The Schiff test invented and named after Hugo Schiff is a chemical test for the detection of aldehydes. An unknown sample is added to the decolorized Schiff reagent and when aldehyde is present a characteristic magenta or purple color develops. The Schiff reagent is the reaction product of...
).
In the 1860s, Christian Friedrich Schönbein
Christian Friedrich Schönbein
Christian Friedrich Schönbein was a German-Swiss chemist who is best known for inventing the fuel cell and his discoveries of guncotton and ozone.- Life :...
and his student Friedrich Goppelsroeder published the first attempts to study the different rates at which different substances move through filter paper. Schönbein, who thought capillary action
Capillary action
Capillary action, or capilarity, is the ability of a liquid to flow against gravity where liquid spontanously rise in a narrow space such as between the hair of a paint-brush, in a thin tube, or in porous material such as paper or in some non-porous material such as liquified carbon fiber, or in a...
(rather than adsorption) was responsible for the movement, called the technique capillary analysis, and Goppelsroeder spent much of his career using capillary analysis to test the movement rates of a wide variety of substances. Unlike modern paper chromatography, capillary analysis used reservoirs of the substance being analyzed, creating overlapping zones of the solution components rather than separate points or bands.
Work on capillary analysis continued, but without much technical development, well into the 20th century. The first significant advances over Goppelsroeder's methods came with the work of Raphael E. Liesegang: in 1927, he placed filter strips in closed containers with atmospheres saturated by solvents, and in 1943 he began using discrete spots of sample adsorbed to filter paper, dipped in pure solvent to achieve separation. This method, essentially identical to modern paper chromatography, was published just before the independent—and far more influential—work of Archer Martin and his collaborators that inaugurated the widespread use of paper chromatography.
Tsvet and column chromatography
The first true chromatography is usually attributed to the Russian botanist Mikhail TsvetMikhail Tsvet
-External links:* * Berichte der Deutschen botanischen Gesellschaft 24, 316–323...
. Tsvet applied his observations with filter paper extraction to the new methods of column fractionation
Fractionation
See also: Fractionated spacecraftFractionation is a separation process in which a certain quantity of a mixture is divided up in a number of smaller quantities in which the composition changes according to a gradient. Fractions are collected based on differences in a specific property of the...
that had been developed in the 1890s for separating the components of petroleum
Petroleum
Petroleum or crude oil is a naturally occurring, flammable liquid consisting of a complex mixture of hydrocarbons of various molecular weights and other liquid organic compounds, that are found in geologic formations beneath the Earth's surface. Petroleum is recovered mostly through oil drilling...
. He used a liquid-adsorption column containing calcium carbonate
Calcium carbonate
Calcium carbonate is a chemical compound with the formula CaCO3. It is a common substance found in rocks in all parts of the world, and is the main component of shells of marine organisms, snails, coal balls, pearls, and eggshells. Calcium carbonate is the active ingredient in agricultural lime,...
to separate yellow, orange, and green plant pigment
Pigment
A pigment is a material that changes the color of reflected or transmitted light as the result of wavelength-selective absorption. This physical process differs from fluorescence, phosphorescence, and other forms of luminescence, in which a material emits light.Many materials selectively absorb...
s (what are known today as xanthophylls, carotenes, and chlorophyll
Chlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
s, respectively). The method was described on December 30, 1901 at the 11th Congress of Naturalists and Doctors (XI съезд естествоиспытателей и врачей) in Saint Petersburg
Saint Petersburg
Saint Petersburg is a city and a federal subject of Russia located on the Neva River at the head of the Gulf of Finland on the Baltic Sea...
. The first printed description was in 1903, in the Proceedings of the Warsaw
Warsaw
Warsaw is the capital and largest city of Poland. It is located on the Vistula River, roughly from the Baltic Sea and from the Carpathian Mountains. Its population in 2010 was estimated at 1,716,855 residents with a greater metropolitan area of 2,631,902 residents, making Warsaw the 10th most...
Society of Naturalists, section of biology. He first used the term chromatography in print in 1906 in his two papers about chlorophyll in the German botanical journal, Berichte der Deutschen Botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for the German Botanical Society. Interestingly, Mikhail's surname "Цвет" means "color" in Russian, so there is the possibility that his naming the procedure chromatography (literally "color writing") was a way that he could make sure that he, a commoner in Tsarist Russia, could be immortalized.
In a 1903 lecture (published in 1905), Tsvet also described using filter paper to approximate the properties of living plant fibers in his experiments on plant pigments—a precursor to paper chromatography
Paper chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it...
. He found that he could extract some pigments (such as orange carotene
Carotene
The term carotene is used for several related unsaturated hydrocarbon substances having the formula C40Hx, which are synthesized by plants but cannot be made by animals. Carotene is an orange photosynthetic pigment important for photosynthesis. Carotenes are all coloured to the human eye...
s and yellow xanthophyll
Xanthophyll
Xanthophylls are yellow pigments that form one of two major divisions of the carotenoid group. The name is from Greek xanthos + phyllon , due to their formation of the yellow band seen in early chromatography of leaf pigments...
s) from leaves with non-polar solvents, but others (such as chlorophyll
Chlorophyll
Chlorophyll is a green pigment found in almost all plants, algae, and cyanobacteria. Its name is derived from the Greek words χλωρος, chloros and φύλλον, phyllon . Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to obtain energy from light...
) required polar solvents. He reasoned that chlorophyll was held to the plant tissue by adsorption
Adsorption
Adsorption is the adhesion of atoms, ions, biomolecules or molecules of gas, liquid, or dissolved solids to a surface. This process creates a film of the adsorbate on the surface of the adsorbent. It differs from absorption, in which a fluid permeates or is dissolved by a liquid or solid...
, and that stronger solvents were necessary to overcome the adsorption. To test this, he applied dissolved pigments to filter paper, allowed the solvent to evaporate, then applied different solvents to see which could extract the pigments from the filter paper. He found the same pattern as from leaf extractions: carotene could be extracted from filter paper using non-polar solvents, but chlorophyll required polar solvents.
Tsvet's work saw little use until the 1930s.
Martin and Synge and partition chromatography
Chromatography methods changed little after Tsvet's work until the explosion of mid-20th century research in new techniques, particularly thanks to the work of Archer John Porter MartinArcher John Porter Martin
Archer John Porter Martin, FRS was a British chemist who shared the 1952 Nobel Prize in Chemistry for the invention of partition chromatography with Richard Synge....
and Richard Laurence Millington Synge. By "the marrying of two techniques, that of chromatography and that of countercurrent solvent extraction", Martin and Synge developed partition chromatography to separate chemicals with only slight differences in partition coefficient
Partition coefficient
In chemistry and the pharmaceutical sciences, a partition- or distribution coefficient is the ratio of concentrations of a compound in the two phases of a mixture of two immiscible solvents at equilibrium. The terms "gas/liquid partition coefficient" and "air/water partition coefficient" are...
s between two liquid solvents. Martin, who had previously been working in vitamin chemistry (including attempts to purify vitamin E
Vitamin E
Vitamin E is used to refer to a group of fat-soluble compounds that include both tocopherols and tocotrienols. There are many different forms of vitamin E, of which γ-tocopherol is the most common in the North American diet. γ-Tocopherol can be found in corn oil, soybean oil, margarine and dressings...
), began collaborating with Synge in 1938, bring his experience with equipment design to Synge's project of separating amino acids. After unsuccessful experiments with complex countercurrent extraction machines and liquid-liquid chromatography methods where the liquids move in opposite directions, Martin hit on the idea of using silica gel in columns to hold water stationary while an organic solvent flows through the column. Martin and Synge demonstrated the potential of the methods by separating amino acids marked in the column by the addition of methyl red
Methyl red
Methyl red, also called C.I. Acid Red 2, is an indicator dye that turns red in acidic solutions. It is an azo dye, and is a dark red crystalline powder....
. In a series of publications beginning in 1941, they described increasingly powerful methods of separating amino acids and other organic chemicals.
In pursuit of better and easier methods of identifying the amino acid constituents of peptides, Martin and Synge turned to other chromatography media as well. A short abstract in 1943 followed by a detailed article in 1944 described the use of filter paper as the stationary phase for performing chromatography on amino acids: paper chromatography
Paper chromatography
Paper chromatography is an analytical chemistry technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary colors in ink experiments. This method has been largely replaced by thin layer chromatography, however it...
. By 1947, Martin, Synge and their collaborators had applied this method (along with Fred Sanger's reagent
Sanger's reagent
1-fluoro-2,4-dinitrobenzene, or Sanger's reagent , is a chemical used for polypeptide sequencing.- Preparation :...
for identifying N-terminal residues) to determine the pentapeptide sequence of Gramicidin S
Gramicidin S
Gramicidin S or Gramicidin Soviet is an antibiotic effective against some Gram positive and Gram negative bacteria as well as some fungi. It is a derivative of gramicidin, produced by the Gram positive bacterium Bacillus brevis...
. These and related paper chromatography methods were also foundational to Fred Sanger's effort to determine the amino acid sequence of insulin
Insulin
Insulin is a hormone central to regulating carbohydrate and fat metabolism in the body. Insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle....
.
Refining the techniques
Martin, in collaboration with Anthony T. James, went on to develop gas chromatography (the principles of which Martin and Synge had laid out in their landmark 1941 paper) beginning in 1949. In 1952, during his lecture for the Nobel Prize in ChemistryNobel Prize in Chemistry
The Nobel Prize in Chemistry is awarded annually by the Royal Swedish Academy of Sciences to scientists in the various fields of chemistry. It is one of the five Nobel Prizes established by the will of Alfred Nobel in 1895, awarded for outstanding contributions in chemistry, physics, literature,...
(shared with Synge, for their earlier chromatography work) Martin announced the successful separation of a wide variety of natural compounds by gas chromatography. (German chemist Fritz Prior had achieved limited success with gas chromatography, separating oxygen
Oxygen
Oxygen is the element with atomic number 8 and represented by the symbol O. Its name derives from the Greek roots ὀξύς and -γενής , because at the time of naming, it was mistakenly thought that all acids required oxygen in their composition...
and carbon dioxide
Carbon dioxide
Carbon dioxide is a naturally occurring chemical compound composed of two oxygen atoms covalently bonded to a single carbon atom...
, in 1947 during his Ph.D. research. The method of Martin and James, however, became the basis for subsequent developments in gas chromatography.)
The ease and efficiency of gas chromatography for separating organic chemicals spurred the rapid adoption of the method, as well as the rapid development of new detection methods for analyzing the output. The thermal conductivity detector
Thermal conductivity detector
The thermal conductivity detector , also known as a Katharometer, is a bulk property detector and a chemical specific detector commonly used in gas-liquid chromatography. This detector senses changes in the thermal conductivity of the column effluent and compares it to a reference flow of carrier gas...
, described in 1954 by N. H. Ray, was the foundation for several other methods: the flame ionization detector
Flame ionization detector
A flame ionization detector is a type of gas detector used in gas chromatography. The first flame ionization detector was developed in 1957 by scientists working for the CSIRO in Melbourne, Australia....
was described by J. Harley, W. Nel, and V. Pretorius in 1958, and James Lovelock
James Lovelock
James Lovelock, CH, CBE, FRS is an independent scientist, environmentalist and futurologist who lives in Devon, England. He is best known for proposing the Gaia hypothesis, which postulates that the biosphere is a self-regulating entity with the capacity to keep our planet healthy by controlling...
introduced the electron capture detector
Electron capture detector
An electron capture detector is a device for detecting atoms and molecules in a gas through the attachment of electrons via electron capture ionization. The device was invented in 1957 by Dr. James E...
that year as well. Others introduced mass spectrometers to gas chromatography in the late 1950s.
The work of Martin and Synge also set the stage for high performance liquid chromatography
High performance liquid chromatography
High-performance liquid chromatography , HPLC, is a chromatographic technique that can separate a mixture of compounds and is used in biochemistry and analytical chemistry to identify, quantify and purify the individual components of the mixture.HPLC typically utilizes different types of stationary...
, suggesting that small sorbent particles and pressure could produce fast liquid chromatography techniques. This became widely practical by the late 1960s (and the method was used to separate amino acids as early as 1960).
Thin layer chromatography
The first developments in thin layer chromatographyThin layer chromatography
Thin layer chromatography is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide, or cellulose...
occurred in the 1940s, and techniques advanced rapidly in the 1950s after the introduction of relatively large plates and relatively stable materials for sorbent
Sorbent
A sorbent is a material used to absorb liquids or gases. Examples include:*A material similar to molecular sieve material. It has a large internal surface area and good thermal conductivity. It is typically supplied in pellets of 1 mm to 2 mm diameter and roughly 5 mm length or as...
layers.
Later developments
In 1987 Pedro CuatrecasasPedro Cuatrecasas
Pedro Cuatrecasas is an American biochemist and an Adjunct Professor of Pharmacology & Medicine at the University of California, San Diego.- Birth and education :...
and Meir Wilchek
Meir Wilchek
Meir Wilchek is an Israeli biochemist.He is a professor at the Weizmann Institute of Science.-Early life and education:Meir Wilchek was born in Warsaw, Poland, scion of a rabbinical family...
were awarded the Wolf Prize in Medicine
Wolf Prize in Medicine
The Wolf Prize in Medicine is awarded once a year by the Wolf Foundation in Israel. It is one of the six Wolf Prizes established by the Foundation and awarded since 1978; the others are in Agriculture, Chemistry, Mathematics, Physics and Arts. The Prize is probably the third most prestigious award...
for the invention and development of affinity chromatography
Affinity chromatography
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.-Uses:Affinity chromatography can be used to:...
and its applications to biomedical sciences.