History Of Chromatography
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The history of chromatography spans from the mid-19th century to the 21st.
Chromatography In chemical analysis, chromatography is a laboratory technique for the Separation process, separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it ...
, literally "color writing", was used—and named— in the first decade of the 20th century, primarily for the separation of plant
pigments A pigment is a powder used to add or alter color or change visual appearance. Pigments are completely or nearly solubility, insoluble and reactivity (chemistry), chemically unreactive in water or another medium; in contrast, dyes are colored sub ...
such as
chlorophyll Chlorophyll is any of several related green pigments found in cyanobacteria and in the chloroplasts of algae and plants. Its name is derived from the Greek words (, "pale green") and (, "leaf"). Chlorophyll allows plants to absorb energy ...
(which is green) and
carotenoid Carotenoids () are yellow, orange, and red organic pigments that are produced by plants and algae, as well as several bacteria, archaea, and fungi. Carotenoids give the characteristic color to pumpkins, carrots, parsnips, corn, tomatoes, cana ...
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 A separation process is a method that converts a mixture or a solution of chemical substances into two or more distinct product mixtures, a scientific process of separating two or more substances in order to obtain purity. At least one product mi ...
es and
chemical analysis Analytical chemistry studies and uses instruments and methods to separate, identify, and quantify matter. In practice, separation, identification or quantification may constitute the entire analysis or be combined with another method. Separa ...
tasks, especially in
biochemistry Biochemistry, or biological chemistry, is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology, a ...
.


Precursors

The earliest use of chromatography—passing a mixture through an inert material to create separation of the solution components based on differential
adsorption Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a ...
—is sometimes attributed to German chemist
Friedlieb Ferdinand Runge Friedlieb Ferdinand Runge (8 February 1794 – 25 March 1867) was a German analytical chemist. Runge identified the mydriatic (pupil dilating) effects of belladonna (deadly nightshade) extract, identified caffeine, and discovered the first ...
, who in 1855 described the use of paper to analyze
dye Juan de Guillebon, better known by his stage name DyE, is a French musician. He is known for the music video of the single "Fantasy Fantasy is a genre of speculative fiction that involves supernatural or Magic (supernatural), magical ele ...
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 : The Schiff test is an early organic chemistry named reaction developed by Hugo Schiff, and is a relatively general chemical test for detection of many organic aldehydes that has also found use in the staining of biological tissues. The Schiff ...
). In the 1860s,
Christian Friedrich Schönbein Christian Friedrich Schönbein HFRSE (18 October 1799 – 29 August 1868) was a German-Swiss chemist who is best known for inventing the fuel cell (1838) at the same time as William Robert Grove and his discoveries of guncotton and ozone. He a ...
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 (sometimes called capillarity, capillary motion, capillary rise, capillary effect, or wicking) is the process of a liquid flowing in a narrow space without the assistance of external forces like Gravitation, gravity. The effe ...
(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 Archer John Porter Martin (1 March 1910 – 28 July 2002) was a British chemist who shared the 1952 Nobel Prize in Chemistry for the invention of partition chromatography with Richard Synge. Early life Martin's father was a GP. Martin was ...
and his collaborators that inaugurated the widespread use of paper chromatography. In 1897, the American chemist David Talbot Day (1859–1915), then serving with the U.S. Geological Survey, observed that crude petroleum generated bands of color as it seeped upwards through finely divided clay or limestone. In 1900, he reported his findings at the First International Petroleum Congress in Paris, where they created a sensation.


Tsvet and column chromatography

The first true chromatography is usually attributed to the Russian-Italian botanist
Mikhail Tsvet Mikhail Semyonovich Tsvet, also spelt Tsvett, Tswett, Tswet, Zwet, and Cvet (Russian: Михаил Семёнович Цвет; 14 May 1872 – 26 June 1919) was a Russian-Italian botanist who invented chromatography. His last name is Russian fo ...
. Tsvet applied his observations with filter paper extraction to the new methods of column
fractionation Fractionation is a separation process in which a certain quantity of a mixture (of gasses, solids, liquids, enzymes, or isotopes, or a suspension) is divided during a phase transition, into a number of smaller quantities (fractions) in which t ...
that had been developed in the 1890s for separating the components of
petroleum Petroleum, also known as crude oil or simply oil, is a naturally occurring, yellowish-black liquid chemical mixture found in geological formations, consisting mainly of hydrocarbons. The term ''petroleum'' refers both to naturally occurring un ...
. He used a liquid-adsorption column containing
calcium carbonate Calcium carbonate is a chemical compound with the chemical formula . It is a common substance found in Rock (geology), rocks as the minerals calcite and aragonite, most notably in chalk and limestone, eggshells, gastropod shells, shellfish skel ...
to separate yellow, orange, and green plant
pigment A pigment is a powder used to add or alter color or change visual appearance. Pigments are completely or nearly solubility, insoluble and reactivity (chemistry), chemically unreactive in water or another medium; in contrast, dyes are colored sub ...
s (what are known today as
xanthophylls Xanthophylls (originally phylloxanthins) are yellow pigments that occur widely in nature and form one of two major divisions of the carotenoid group; the other division is formed by the carotenes. The name is from Greek: (), meaning "yellow", an ...
,
carotenes The term carotene (also carotin, from the Latin ''carota'', "carrot") is used for many related unsaturated hydrocarbon substances having the formula C40Hx, which are synthesized by plants but in general cannot be made by animals (with the e ...
, and
chlorophyll Chlorophyll is any of several related green pigments found in cyanobacteria and in the chloroplasts of algae and plants. Its name is derived from the Greek words (, "pale green") and (, "leaf"). Chlorophyll allows plants to absorb energy ...
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, formerly known as Petrograd and later Leningrad, is the List of cities and towns in Russia by population, second-largest city in Russia after Moscow. It is situated on the Neva, River Neva, at the head of the Gulf of Finland ...
. The first printed description was in 1903, in the Proceedings of the
Warsaw Warsaw, officially the Capital City of Warsaw, is the capital and List of cities and towns in Poland, largest city of Poland. The metropolis stands on the Vistula, River Vistula in east-central Poland. Its population is officially estimated at ...
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. 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 is an analytical method used to separate colored chemicals or substances. It can also be used for colorless chemicals that can be located by a stain or other visualisation method after separation. It is now primarily used as ...
. He found that he could extract some pigments (such as orange
carotene The term carotene (also carotin, from the Latin ''carota'', "carrot") is used for many related unsaturated hydrocarbon substances having the formula C40Hx, which are synthesized by plants but in general cannot be made by animals (with the ex ...
s and yellow
xanthophyll Xanthophylls (originally phylloxanthins) are yellow pigments that occur widely in nature and form one of two major divisions of the carotenoid group; the other division is formed by the carotenes. The name is from Greek: (), meaning "yellow", an ...
s) from leaves with
non-polar solvent A solvent (from the Latin language, Latin ''wikt:solvo#Latin, solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a Solution (chemistry), solution. A solvent is usually a liquid but can also be a solid, a gas ...
s, but others (such as
chlorophyll Chlorophyll is any of several related green pigments found in cyanobacteria and in the chloroplasts of algae and plants. Its name is derived from the Greek words (, "pale green") and (, "leaf"). Chlorophyll allows plants to absorb energy ...
) required
polar solvent A solvent (from the Latin language, Latin ''wikt:solvo#Latin, solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a Solution (chemistry), solution. A solvent is usually a liquid but can also be a solid, a gas ...
s. He reasoned that chlorophyll was held to the plant tissue by
adsorption Adsorption is the adhesion of atoms, ions or molecules from a gas, liquid or dissolved solid to a surface. This process creates a film of the ''adsorbate'' on the surface of the ''adsorbent''. This process differs from absorption, in which a ...
, 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 Martin Archer John Porter Martin (1 March 1910 – 28 July 2002) was a British chemist who shared the 1952 Nobel Prize in Chemistry for the invention of partition chromatography with Richard Synge. Early life Martin's father was a GP. Martin was e ...
and
Richard Laurence Millington Synge Richard Laurence Millington Synge (28 October 1914 – 18 August 1994) was a British biochemist, and shared the 1952 Nobel Prize in Chemistry for the invention of partition chromatography with Archer Martin. Life Richard Laurence Millington ...
. By "the marrying of two techniques, that of chromatography and that of countercurrent solvent extraction", Martin and Synge developed
partition chromatography Partition chromatography theory and practice was introduced through the work and publications of Archer Martin and Richard Laurence Millington Synge during the 1940s. They would later receive the 1952 Nobel Prize in Chemistry "for their invention ...
to separate chemicals with only slight differences in
partition coefficient In the physical sciences, a partition coefficient (''P'') or distribution coefficient (''D'') is the ratio of concentrations of a chemical compound, compound in a mixture of two immiscible solvents at partition equilibrium, equilibrium. This rati ...
s between two liquid solvents. Martin, who had previously been working in vitamin chemistry (including attempts to purify
vitamin E Vitamin E is a group of eight compounds related in molecular structure that includes four tocopherols and four tocotrienols. The tocopherols function as fat-soluble antioxidants which may help protect cell membranes from reactive oxygen speci ...
), began collaborating with Synge in 1938, brought his experience with equipment design to Synge's project of separating
amino acids Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the Proteinogenic amino acid, 22 α-amino acids incorporated into p ...
. 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 (2-(''N'',''N''-dimethyl-4-aminophenyl) azobenzenecarboxylic acid), 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. Methyl red is a pH indica ...
. 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 also turned to other chromatography media. 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 is an analytical method used to separate colored chemicals or substances. It can also be used for colorless chemicals that can be located by a stain or other visualisation method after separation. It is now primarily used as ...
. By 1947, Martin, Synge and their collaborators had applied this method (along with Fred Sanger's reagent for identifying
N-terminal The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
residues) to determine the pentapeptide sequence of
Gramicidin S Gramicidin S or Gramicidin Soviet is an antibiotic that is 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 ''Brevibacillus brevis ...
. These and related paper chromatography methods were also foundational to
Fred Sanger Frederick Sanger (; 13 August 1918 – 19 November 2013) was a British biochemist who received the Nobel Prize in Chemistry twice. He won the 1958 Chemistry Prize for determining the amino acid sequence of insulin and numerous other prot ...
's effort to determine the
amino acid sequence Protein primary structure is the linear sequence of amino acids in a peptide or protein. By convention, the primary structure of a protein is reported starting from the amino-terminal (N) end to the carboxyl-terminal (C) end. Protein biosynthe ...
of
insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the insulin (''INS)'' gene. It is the main Anabolism, anabolic hormone of the body. It regulates the metabol ...
.


Refining the techniques

Martin, in collaboration with Anthony T. James, went on to develop
gas chromatography Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for Separation process, separating and analyzing compounds that can be vaporized without Chemical decomposition, decomposition. Typical uses of GC include t ...
(GC; the principles of which Martin and Synge had predicted in their landmark 1941 paper) beginning in 1949. In 1952, during his lecture for the
Nobel 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 outst ...
(shared with Synge, for their earlier chromatography work), Martin announced the successful separation of a wide variety of natural compounds by gas chromatography. Previously,
Erika Cremer Erika Cremer (20 May 1900, Munich – 21 September 1996, Innsbruck) was a German physical chemist and Professor Emeritus at the University of Innsbruck who is regarded as one of the most important pioneers in gas chromatography, as she second c ...
had laid the theoretical basis of GC in 1944 and Austrian chemist Fritz Prior, under the direction of Erika Cremer, constructed in 1947 the first prototype of a gas chromatograph and achieved separating
oxygen Oxygen is a chemical element; it has chemical symbol, symbol O and atomic number 8. It is a member of the chalcogen group (periodic table), group in the periodic table, a highly reactivity (chemistry), reactive nonmetal (chemistry), non ...
and
carbon dioxide Carbon dioxide is a chemical compound with the chemical formula . It is made up of molecules that each have one carbon atom covalent bond, covalently double bonded to two oxygen atoms. It is found in a gas state at room temperature and at norma ...
, in 1947 during his Ph.D. research. 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 The thermal conductivity detector (TCD), also known as a katharometer, is a bulk property detector and a chemical specific detector commonly used in gas chromatography. This detector senses changes in the thermal conductivity of the column eluent a ...
, described in 1954 by N. H. Ray, was the foundation for several other methods: the
flame ionization detector A flame ionization detector (FID) is a scientific instrument that measures analytes in a gas stream. It is frequently used as a detector in gas chromatography. The measurement of ions per unit time makes this a mass sensitive instrument. Standa ...
was described by J. Harley, W. Nel, and V. Pretorius in 1958, and
James Lovelock James Ephraim Lovelock (26 July 1919 – 26 July 2022) was an English independent scientist, environmentalist and futurist. He is best known for proposing the Gaia hypothesis, which postulates that the Earth functions as a self-regulating syst ...
introduced the
electron capture detector An electron capture detector (ECD) 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 James Lovelock and is used in gas chromatography to ...
that year as well. Others introduced
mass spectrometer Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is us ...
s 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 (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify specific components in mixtures. The mixtures can origina ...
, 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 chromatography Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures. It is performed on a TLC plate made up of a non-reactive solid coated with a thin layer of adsorbent material. This is called the sta ...
occurred in the 1940s, and techniques advanced rapidly in the 1950s after the introduction of relatively large plates and relatively stable materials for
sorbent A sorbent is an insoluble material that either absorbs or adsorbs liquids or gases. They are frequently used to remove pollutants and in the cleanup of chemical accidents and oil spills. Besides their uses in industry, sorbents are used in comm ...
layers. Touchstone, pp. 1651–1652


Later developments

In 1987
Pedro Cuatrecasas Pedro Cuatrecasas (27 September 1936 – 19 March 2025) was a Spanish-born American biochemist and academic who was an adjunct professor of Pharmacology & Medicine at the University of California San Diego. Background Pedro Cuatrecasas was b ...
and
Meir Wilchek Meir Wilchek (; born 17 October 1935) 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. During the Holocaust, he esca ...
were awarded the
Wolf Prize in Medicine The Wolf Prize in Medicine is awarded annually 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 ...
for the invention and development of
affinity chromatography Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the ...
and its applications to biomedical sciences.


References


Cited sources

* * * {{color topics
Chromatography In chemical analysis, chromatography is a laboratory technique for the Separation process, separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it ...
Chromatography In chemical analysis, chromatography is a laboratory technique for the Separation process, separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it ...
Chromatography