Chromatography is a method of physicochemical analysis separating constituents of a mixture (solutes) by entrainment by means of a mobile phase (liquid or gas) along a stationary phase (liquid or solid fixed) through the selective partition solutes between the two phases. Each solute is therefore subject to a retention force (exerted by the stationary phase) and force mobility (due to the mobile phase).
The first chromatography was performed in the beginning 20th century by Mikhail Tswett and was to separate the pigments (Greek: “chromatography”) of a spinach leaf. Tswett separation was observed vegetable dyes, of which the chlorophylls, filtered when their solution in petroleum ether, on a column of calcium carbonate. Under these conditions, in fact, green and yellow colored areas are formed. Tswett stated: “Just as the rays of the spectrum, the different components of a mixture of dyes are deployed on the column of calcium carbonate according to a law and can be analyzed qualitatively and quantitatively.”
This invention adsorption chromatography Tswett remained unknown until 1931, when Kuhn, Winterstein Lederer and exploited it again with success.
The main stages of the evolution of chromatography
1906 – The Russian botanist, Mr. Tswett published his. Book: “The chromophylles in the plant and animal world”, where the method of separation of pigments is described in detail.
1931 – Khun Lederer and separated at a preparative scale carotenes and xanthophylls. The long sleep of the method Tswett is broken and it is growing rapidly, thanks to the work of Brokmann, Karrer, Winterstein and Zechmeister.
1938 – Reichstein introduced “liquid chromatogram” enable colorless substance separations. This form of chromatography has been very widely used.
1940-1943 – Tiselieus develops its methods “frontal analysis” and “development through movement”.
1941 – Martin and Synge introduce partition chromatography on silica gel. Now, instead of a few grams of protein, a few milligrams are sufficient for the analysis of neutral amino acids.
1944 – Consden, Gordon and Martin invented partition chromatography on paper, very ingenious method, to analyze not just a few milligrams, but a few grams of amino acids, sugars, etc.
1940-1947 – Wilson, Devault, Weiss, Gluckauf Martin, Synge and others develop detailed theories of chromatography.
1947 – A group of American researchers, including Boyd, Marinsky, Spedding, Tompkins, etc.., Publish details of their work for the separation of rare earths and radioactive substances on ion exchangers. This research has important separations on an industrial scale and is the basis for the manufacture of certain isotopes currently on the market. Chromatography is thus assured an important place in Inorganic Chemistry.
Different types of chromatographic techniques
Factors involved in the sharing of molecules to be separated between fixed and mobile phases: solubility in a liquid solvent, the size (shape), the polarity of the electric charge, and the presence of atoms forming groupernents sites individuals. Different types of chromatography result from the fact that we have privileged the effect of one of these factors, but an exclusive mechanism is never complete during chromatographic separation.
The mobile phase is a liquid. Depending on the nature of the stationary phase can be distinguished:
- Chromatographies sharing: This is a liquid-liquid chromatography. The stationary phase is a liquid attached to an inert carrier. This chromatography is so named because it is based on the sharing of the solute in the two liquid phases.
- Exclusion chromatography: it is still called diffusion-exclusion chromatography, molecular sieving, gel filtration, gel permeation. The stationary phase is a porous solid: large particles are excluded from the stationary phase, however small embedded particles diffuse into the pores of the gel.
- The adsorption chromatography: This is a liquid-solid chromatography. The stationary phase is a solid adsorbent polar. Adsorption chromatography is a reverse phase liquid-solid chromatography in which the stationary phase is nonpolar.
- The ion exchange chromatography: The stationary phase is an ion exchanger consisting of a carrier resin groups ionized either negatively or positively, exerting electrostatic interactions with the medium ionic solutes.
- Affinity chromatography: The stationary phase is a chemically inert macromolecular carrier, onto which is grafted an effector which has a biological affinity (bio-affinity) to a solute of the sample (enzyme-substrate affinity, ligand-receptor , antigen-antibody).
The mobile phase is a carrier gas. We distinguish in this case:
- The gas-liquid chromatography: This is a partition chromatography. The stationary phase is a liquid fixed by imbibition of an inert carrier.
- The gas-solid chromatography: This is an adsorption chromatography. The stationary phase is a solid adsorbent.
Different chromatographic principles result from the fact that we have privileged the effect of the following factors, in order to separate a mixture of compounds comprising:
- Solubility in a solvent in liquid partition chromatography
- The size, shape, size-exclusion chromatography in
- Polarity in adsorption chromatography and reverse phase adsorption
- The electric charge in the ion exchange chromatography
- The presence of atoms forming groupernents of specific sites in the affinity chromatography