Chromatography
 
Chromatography constitutes a family of closely related methods for separating and analyzing a wide variety of chemicals. Today, despite developments in analytical chemistry that link scientists to many modern and extremely sophisticated devices, the classic methodology of chromatography still plays a very important role among analytical techniques. It is almost impossible to imagine a laboratory without chromatographic equipment. Quality control, product purification, and basic research are some of the fields in which chromatography is used.
1. Definition
The term chromatography, first used by Tsvet (1872–1919), a Russian botanist, derived from the two Greek words Khromatos (color) and graphos (written). He used the term chromatography to describe his studies on pigment separation using a chalk column (1, 2), thus defining chromatography as a method by which the components of a mixture were separated on an adsorbent column in a flowing system (1, 2). The International Union of Pure and Applied Chemistry (IUPAC) has further defined chromatography as  A method, used primarily for separation of the components of a sample, in which the components are distributed between two phases, one of which is stationary while the other moves. The stationary phase may be a solid, or a liquid supported on a solid, or a gel. The stationary phase may be packed in a column, spread as a layer , or distributed as a film, etc.; in these definitions chromatographic bed is used as a general term to denote any of the different forms in which the stationary phase may be used. The mobile phase may be gaseous or liquid (3).
2. Historical Perspective
Although some phenomena that form the basis of chromatographic methods have been known for a long time, Tsvet is generally referred to as the father of chromatography. In 1906, he described the separation of plant pigments by column liquid chromatography using over 100 adsorption media (1, 4, 5 ،). During the next forty years after Tsvet's work, there were some important developments in the field. For example, Kuhn et al. published two papers in 1931 on separating carotenoids on a calcium carbonate column (6, 7). Kuhn, Karrer, and Ruzicka applied the chromatographic technique to their own fields of interest and were awarded the Nobel prize (1937, 1938, and 1939, respectively) for their contributions to chromatography. Tiselius (8) and Claesson (9) developed the now classical procedures involving the continuous observation of optical properties of solutions flowing out of chromatographic columns. Tiselius was awarded the Nobel prize in 1948 for his research on “Electrophoresis and Adsorption Analysis”. The introduction of gradient elution in 1952 (10) was an important contribution to all column chromatographic methods.
 Chromatograpic developments greatly accelerated after the famous paper by Martin and Synge appeared in 1941 (11). They presented the invention of liquid-liquid (or partition) chromatography in columns and in planar form (paper chromatography). They also provided a theoretical framework for the basic chromatographic process. Martin and Synge were awarded the Nobel prize in 1952 for this work. When thin layers of supported silica gel were introduced as an alternative for paper in the late 1950s (12, 13), the field of thin-layer chromatography (TLC) was born and became so popular that it has largely replaced the older technique. Another main development in the progress of chromatography was the introduction of gas-liquid chromatography (GLC) by James and Martin (14) , which had an unprecedented impact on the analytical chemistry of organic compounds. Porath and Flodin introduced size-exclusion chromatography in 1959 (15), allowing easy separation of macromolecules. Modern liquid chromatography (HPLC) was introduced in the early 1970s, permitting the efficient separation of a wide range of components.
The theory of chromatography was first studied by Wilson (16), who discussed the quantitative aspects in terms of diffusion, adsorption rate, and isothermal nonlinearity. The plate theory was first presented by Martin and Synge (11) and was further explored by Craig (17) and Gluechauf (18). In this theory, chromatography is described in terms closely analogous in its mode of operation to distillation and extraction fractionating columns. Lapidus and Admunson (19), followed by van Deemter and co-workers (20), developed the rate theory, an alternative to the plate theory. In this theory, column efficiency was described as a function of the mobile-phase's flow rate and diffusion properties and the stationary-phase particle size. In 1959, Giddings published another paper on this topic (21), and the rate theory has since become the backbone of chromatographic theory. In 1963, Giddings (22) pointed out that, if the efficiencies of gas chromatography were to be achieved in liquid chromatography, particle sizes of 2 to 20 µm were required. This prediction was found to be correct with the development of HPLC systems. Numerous detailed descriptions of chromatographic theory exist in the literature. Notable examples include a monograph edited by Jönsson (23) and the excellent reviews of Snyder (24) and Horváth and Melander (25).
This is only a small glimpse of the historical development of chromatography. The book 75 Years of Chromatography—A Historical Dialogue (26), which describes many of the individuals who deserve credit for developing this technique, can be consulted for more complete accounts.
 3.  Retention Mechanism Classification
There are three common ways to classify chromatographic methods. The first and most popular classification is based on the mechanisms of retention, the manner in which the analyte interacts with the stationary phase. In this classification, chromatography may be divided into the five following basic types:
3.1. Adsorption Chromatography 
This technique is based on competition for neutral analytes between the mobile phase (gas or liquid( and a neutral solid adsorbent. Therefore, analytes with polar groups are retained longer by a polar adsorbent, and nonpolar analytes interact better with a nonpolar stationary phase. In this type of chromatography, the analytes are simultaneously in contact with both the stationary phase and the mobile phase.
3.2. Partition Chromatography 
This technique is based on competition for neutral analytes between the mobile phase (gas or liquid(  and a neutral liquid or liquid-like stationary phase (the latter is usually called a “bonded-phase” when long alkyl chains or their derivatives are bonded to a matrix and behave like a liquid). In partition chromatography, the analyte is transferred from the bulk of one phase into the bulk of the other, so that the analyte molecules are surrounded only by molecules of one phase. Separation in partition chromatography is achieved by differences in the partition coefficients of the analytes between the mobile and stationary phases.
3.3. Ion-Exchange Chromatography 
This technique is based on the electrostatic interaction between a charged solute and an oppositely charged solid stationary phase. Separation in ion-exchange chromatography is achieved by the differing affinities of ions in solution for oppositely charged ionic groups in the stationary phase. Ion-exchange chromatography is applicable to any solute that acquires a charge in solution. Thus, even carbohydrates, which are largely uncharged below pH 12, are separated by this type of chromatography at sufficiently high pH.
3.4. Size-Exclusion Chromatography 
This technique is based on the sieving principle and is variously known as gel chromatography, gel filtration and gel-permeation chromatography. In this technique, the stationary-phase particles have a wide range of pore sizes, causing the stationary phase to behave like a molecular sieve. Small molecules permeate the pores, and large bulky molecules are excluded. Thus, the solutes are separated on the basis of molecular weight and size, and the larger ones elute first.
3.5. Affinity Chromatography 
This technique is based on the unique biological specificity of ligand-binding interactions (ie, the lock-and-key mechanism). The ligand is covalently bonded to the matrix that forms packing material for the column. Separation is achieved after the applied macromolecule becomes specifically, but not irreversibly, bound to the ligand. The macromolecule is eluted by altering the composition or pH of the eluent so as to weaken its interaction with the ligand, thus promoting dissociation and facilitating elution of the retained compounds.
Many other types of chromatography, such as hydrophobic-interaction, chiral, ion-pair, and salting-out chromatography, are also frequently used. Furthermore, in practical chromatography intermediate or mixed types are often used. So, although one dominant mechanism is presented, the chromatographic modes are not mutually exclusive. To know more about the relationships of different chromatographic modes, see a schematic diagram by Saunders (27).
 4. Development Procedure Classification
The second classification is based on the development procedure, the mechanism by which the sample is removed from the column, and therefore depends on the nature of the mobile phase. This classification was introduced by Tiselius (8) in 1940. There are three chromatographic modes in the classification: (1) elution development, (2) displacement development, and (3) frontal analysis. The principles of each mode are illustrated by Braithwaite and Smith (28). In practice, only elution development and to a lesser extent displacement development are commonly used.
5. Fractionation Phase Classification
The third classification is based on the phases between which the fractionation process takes place. In chromatography, one phase is held immobile or stationary, and the other one (the mobile phase) is passed over it. Therefore chromatography is mainly divided into two large groups named according to the state of aggregation of the mobile phase, liquid chromatography and gas chromatography. Further groupings can be made by naming both the mobile and stationary phases, thus liquid-liquid, liquid-solid , gas-liquid, and gas-solid chromatography have been named. More recently, supercritical fluids have been used as mobile phases, and these techniques have been named supercritical fluid chromatography, irrespective of the state of the stationary phase.
References
1. M. S. Tsvet (1906) Ber. Deut. Botan. Ges. 24, 316. 
2. M. S. Tsvet (1906) Ber. Deut. Botan. Ges. 24, 384. 
3. Recommendations on Nomenclature for chromatography, Rules Approved 1973, IUPAC Analytical Chemistry Division Commission on Analytical Nomenclature (1974) Pure Appl. Chem. 37, 447. 
4.M. S. Tsvet (1910) Khromofilly v Rastitel''nom i Zhivotnom Mire Tipogr. Varshavskago Uchebnago Okruga, Warsaw. 
5.H. H. Strain and J. Sherma (1967) J. Chem. Educ. 44, 238–242. 
6.R. Kuhn and E. Lederer (1931) Ber. 64, 1349. 
7. R. Kuhn, A. Winterstein, and E. Lederer (1931) Hoppe Seyler''s Z. Physiol. Chem. 197, 141–160.
8. A. Tiselius (1940) Ark. Kemi. Mineral. Geol. 14B, 22. 
9. S. Claesson (1946) Ark. Kemi. Mineral. Geol. 23A, 1. 
10. R. S. Alm, R. J. P. Williams, and A. Tiselius (1952) Acta Chem. Scand. 6, 826–836. 
11. A. J. P. Martin and R. L. M. Synge (1941) Biochem. J. 35, 1358–1368. 
12. E. Stahl et al. (1956) Pharmazie 11, 633. 
13. E. Stahl (ed.) (1962) Thin Layer Chromatography, Academic Press, New York. 
14. A. T. James and A. J. P. Martin (1952) Biochem. J. 50, 679–690. 
15. J. Porath and P. Flodin (1959) Nature 183, 1657–1659. 
16. J. N. Wilson (1940) J. Am. Chem. Soc. 62, 1583–1591. 
17. L. C. Craig (1950) Anal. Chem. 22, 1346–1352. 
18. E. Glueckauf (1955) Trans. Faraday Soc. 51, 34–44. 
19. L. Lapidus and N. R. Amundson (1952) J. Phys. Chem. 56, 984–988.
20. J. J. van Deemter, F. J. Zuiderweg, and A. Klinkenberg (1956) Chem. Eng. Sci. 5, 271. 
21.  J. C. Giddings (1959) J. Chem. Phys. 31, 1462—1467. 
22.  J. C. Giddings (1963) Anal. Chem. 35, 2215–2216. 
23.J. Å. Jönsson (ed.) (1987) Chromatographic Theory and Basic Principles (Chromatographic Science Series 38), Marcel Dekker, New York. 
24. L. R. Snyder (1992) in Chromatography (E. Heftmann, ed) (J. Chromatogr. Library, Vol. 51A,( Elsevier, Amsterdam, pp. A1–A68. 
25. C. Horváth and W. R. Melander (1983) in Chromatography (E. Heftmann, ed.) (J. Chromatogr. Library, Vol. 22A), Elsevier, Amsterdam, pp. A27–A135. 
26. L. S. Ettre and A. Zlatkis (1979) 75 Years of Chromatography-A Historical Dialogue (L.S. Ettre and A. Zlatkis, eds.) (J. Chromatogr. Library, Vol. 17), Elsevier, Amsterdam. 
27. D. L. Saunders (1975) in Chromatography, 3rd ed. (E. Heftmann, ed), Van Nostrand Reinhold, New York, p. 81. 
28. A. Braithwaite and F. J. Smith (1985) Chromatographic Methods, 4th ed., Chapman and Hall, New York, p. 8.