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The Way We Were CREATING A CENTRAL SCIENCEA brief history of "color writing"Mark S. LesneyC hromatography is one of the most truly chemical methods for manipulating and understanding the natural world. It is an enabling technology for a wide range of products and discoveries, including those of more than a half-dozen Nobel Prize winners. All of the various forms of chromatography rely on differential solubility or adsorption of compounds to separate molecules between a stationary phase and a mobile phase (Figure 1; Table 1).
The history of chromatography is a history of discovery - of new phases, new methods of detection, and new applications for old techniques. From the first use of filter paper to examine plant pigments in the mid-nineteenth century to the development of the most modern high-pressure columns and affinity gels, today chromatography continues as one of the fundamental tools of modern biotechnology, providing much of the technological justification behind chemistry's claim of being "the central science." Despite several earlier false starts (see sidebar, The "What If?" Game), chromatography is a technology of the twentieth century. In 1903, the Russian botanist Mikhail Semenovich Tswett (considered the father of chromatography for coining the term, from the Latin for "color writing") first reported the process of column adsorption chromatography. He passed extracts of plant tissue through a chalk column to separate pigments by differential adsorption. His results, and especially his technique, were derided by rival scientists whose own findings and methodologies he attacked. His most significant opponent was R. M. Willstätter, the "Pope" of German chemistry, who went on to win the Nobel Prize in 1915 for analyzing chlorophyll and other plant pigments. Willstätter presented many of the same results (using different methods) that Tswett had gotten nearly ten years before. To make matters worse, because Tswett published in Russian, his work was less available to the scientific community than that of his detractors.
In 1922, L. S. Palmer, an American scientist, used Tswett's technique on various natural products, but it wasn't until 1931 that Richard Kuhn (one of Willstätter's students) and others used chromatography to separate isomers of polyene pigments, promoting acceptance of the method. Referring to these breakthroughs, traditional chemist and Nobel Laureate Heinrich Wieland complained: "Up to now, we have learned with much effort to distill, crystallize, and recrystallize, and now they come along and just pour the stuff through a little tube!" THE MAIN TECHNIQUESLiquid-liquid partition (column) chromatography. In 1941, Archer John Porter Martin and Richard Laurence Millington Synge, working for the Wool Industries Research Association in England, created liquid-liquid partition chromatography. It was arguably the most significant tool for linking analytical chemistry to the life sciences; it would help create and define molecular biology research. Partition chromatography was developed to separate the various amino acids that defined proteins. Martin had originally used chains of separating funnels to move upper and lower liquid layers for countercurrent extraction, but even with just a few sets of funnels, shaking and separating the layers became a full-time job. The key innovation in developing liquid-liquid partition chromatography was to use a solid support, which made it possible to create a liquid stationary phase. In 1940, Martin and Synge ground up the silica gel being used as a drying agent inside a balance case, added water, put it in a column, and poured chloroform plus their amino acid mixture through it. They added methyl orange stain and found that the amino acids passing down the column appeared as red bands. This was the foundation of partition chromatography for which they would be given the Nobel Prize in 1952.
Paper chromatography. Rather than resting on his laurels, Martin (with his colleagues R. Consden and A. H. Gordon) went on to create paper chromatography in 1944 - one of the most important methods in the development of biotechnology. In paper chromatography, the paper acts as a flat "column," providing the solid phase on which a stationary polar solvent and a mobile, less polar solvent could interact. When, as shown in Figure 2, the original chromatogram was turned ninety degrees, a second solvent partitioning could be performed, allowing separations in two dimensions. This enabled the routine isolation and identification of amino acids and nucleic acids unattainable by column chromatography. It was one of the key techniques (along with ion-exchange chromatography) that led to Frederick Sanger's 1958 Nobel Prize for determining the first amino acid sequence of a protein, insulin. Today, paper chromatography has largely been replaced by thin-layer chromatography and various forms of electrophoresis.
Gas chromatography (GC and GLC). In November 1945, the first analytical gas-solid (adsorption) chromatograph was developed in the chaos of post-war Germany by Fritz Prior, a graduate student under the direction of Erika Cremer, then head of the Institute of Physical Chemistry at Innsbruck University. By early spring 1947, Prior succeeded in separating oxygen and carbon dioxide on a charcoal column - a technical achievement for which he received his Ph.D. Then, in 1950 in England, still not content to rest on previous discoveries, Martin, with yet another colleague, Anthony T. James, developed gas-liquid (partition) chromatography (GLC). Although Martin and Synge had predicted GLC in their original paper on liquid-liquid chromatography in 1941, no one had taken them up on it, so Martin decided to try it himself. He and James used the new column with great success to separate a variety of natural products. Martin casually announced the development of the method in his Nobel Prize lecture in 1952.
Today, except for specialized applications, GLC is the method of choice in countless laboratories, and many scientists claim that it is the most widely applied analytical technique in the world. Originally, packed columns were used exclusively, but because of the work of M.J.E. Golay, as well as Leslie Ettre and co-workers at Perkin-Elmer, the so-called SCOT (support-coated open tubular) capillary columns were designed to allow for extremely large chromatographic efficiencies in small volumes across great column lengths. The use of small sample volumes both required and permitted the development of more sophis- ticated detector apparatus, including various emission and ionization detectors, which replaced the early thermal detectors. The utility of GC quickly led to a host of combined analysis techniques, such as GC-IR and GC/MS (both first coupled in the mid-1950s), which are still popular today. Commercially available gas chromatographs were produced in 1955 by Burrell Corp., Perkin-Elmer, and Podbielniak. Thin-layer chromatography (TLC). In 1938, Russian scientists N. A. Izmailov and M. S. Shraiber developed drop chromatography on horizontal thin layers. After World War II, two American chemists, J. E. Meinhard and N. F. Hall, used it to separate terpenes found in essential oils. Inspired by their work, Justus G. Kirchner and his co-workers at the US Department of Agriculture's Fruit and Vegetable Laboratory in California perfected TLC throughout the early 1950s. Silicic acid with a starch binder was applied to glass plates to support ethyl acetate as the stationary, and hexane as the mobile, liquid phase. It was not until uniformity and availability of equipment (in this case, better coating media and more efficient apparatus for applying them to plates) were guaranteed that the general chemical community adopted the technique. Although profoundly useful in its own right, often TLC serves as a test run of solvent systems for large-scale column chromatography or as a convenient assay tool for column eluants using a variety of visualization techniques. Because of its utility and reproducibility, TLC has practically replaced paper chromatographic methods. As with all forms of chromatography, the choice of coating, whether inert or reactive, depends on the nature of the material to be separated and the end results desired. Recently, TLC has proved especially practical for separating enantiomers through the use of specialized cyclodextrin coatings. This process is the basis for chiral chromatography (also applicable in column form) - a critically important technique for the production of biologically active drugs.
High-pressure liquid chromatography (HPLC). Although begun in 1966, when Csaba Horvàth at Yale University named it, HPLC did not catch on until the early 1970s, when the production of silanized silica yielded a packing material that allowed the use of smaller volume and longer columns with the pressurized solvents. Combined with the introduction of gradient elution in the 1950s by Arne Tiselius and co-workers, the new packing materials proved the basis for HPLC. Gradient elution uses a mixed-solvent mobile phase whose components change in concentration over time, producing a gradient. This process creates a series of new conditions for differential solubility between the stationary and mobile phases. HPLC was popularized throughout the 1970s as a sophisticated improvement over open columns and provided the more precise and rapid separations required for many areas of industrial biotechnology. Typical detection was via UV-absorbance, which was especially useful for protein and nucleic acid determinations. (Coincidentally, HPLC - as with many others, it seems - was predicted in 1941 by that modern Nostradamus of instrumentation, A.J.P. Martin, in his famous paper with R.L.M. Synge.) Ion-exchange chromatography (IEC) and ion chromatography (IC). Ion-exchange methods have been in use since 1850, when H. Thompson and J. T. Way, researchers in England, treated various clays with ammonium sulfate or carbonate in solution to extract the ammonia and release calcium. In 1927, the first zeolite mineral column was used to remove interfering calcium and magnesium ions from solution to determine the sulfate content of water. The modern version of IEC was developed during the wartime Manhattan Project. A technique was required to separate and concentrate the radioactive elements needed to make the atom bomb. Researchers chose adsorbents that would latch onto charged transuranium elements, which could then be differentially eluted. Ultimately, once declassified, these techniques would use new IE resins to develop the systems that are often used today for specific purification of biologicals and inorganics. In the early 1970s, ion chromatography was developed by Hamish Small and co-workers at Dow Chemical Company as a novel method of IEC usable in automated analysis. IC uses weaker ionic resins for its stationary phase and an additional neutralizing stripper, or suppressor, column to remove background eluent ions. It is a powerful technique for determining low concentrations of ions and is especially useful in environmental and water quality studies, among other applications.
OTHER TECHNIQUES Since the 1930s, Tiselius worked on developing electrophoresis as part of US research on human plasma. His techniques proved fundamental to the development of electrochromatography, a catch-all term for any form of chromatography, such as capillary electrophoresis, which uses electromotive force as an aid to separations. In the 1950s, Tiselius and co-workers adapted molecular sieving chromatography - a means of sorting macromolecules by size and molecular weight - for the purification of a host of biologically important compounds, both natural and synthetic. These are just some of the many "minor" forms of chromatographic analysis, each with its own history. Despite the relatively small number of people involved in chromatography's early days, it is difficult to name a facet of modern life to which it has not contributed, whether as an analytical science, a preparative tool, or a regulatory technique - including its role as a mainstay of the chemical industry. In 1990 surveys, gas-liquid chromatographs were the most frequently mentioned analytical instruments for purchase. Chromatography - with its fundamental quest to separate and identify every kind of atom and molecule, and with its diverse applications from agriculture to zoology, from petrochemicals to biopharmaceuticals, and from simple salts to the most complex polymers - can indeed be considered the instrumental backbone of chemistry's claim to be the central science.
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