1 Introduction
Chromatography is a powerful separation method that finds applications in all branch of science. Chromatography was invented and named by the Russian botanist Mikhail Tswett shortly after the turn of the last century. He employed this technique to separate various plant pigments such as chlorophylls and xanthophylls by passing solutions of these compounds through a glass column packed with finely divided calcium carbonate. The separated species appeared as colored bands on the column, which accounts for the name he chose for the method (Greek chroma meaning color and graphein meaning writing).The application of chromatography have grown explosively in the last half century, due not only to the development of several new types of chromatographic techniques but also to the growing need by scientists for better methods for characterizing complex mixtures. The tremendous impact of these methods on sciences is attested by the 1952 Nobel Prize in chemistry that was awarded to A.J.P.Martin and R.L.M.Synge for their discoveries in the field.
Chromatography involves a sample or sample extract being dissolved in a mobile phase, which may be a gas, a liquid or a supercritical fluid. The mobile phase is then forced through an immobile, immiscible stationary phase, which is fixed in place in a column or on a solid surface. The two phases are chosen so that the components of the sample distribute themselves between the mobile and stationary phases to varying degrees. Those components strongly retained by the stationary phase move only slowly with the flow of mobile phase. In contrast, components that are weakly held by the stationary phase travel rapidly. As a consequence of these differences in migration rates, sample components separate into discrete bands, or zones, that can be analyzed qualitatively and quantitatively.1
1.1 Methods in chromatography2
Chromatographic techniques can be classified according to the nature of the stationary and mobile phases as follows:
Adsorption chromatography
• Gas solid chromatography
• Liquid column chromatography
• High performance liquid chromatography
• Thin layer chromatography
Partition chromatography
• Gas solid chromatography
• Super critical fluid chromatography
• Liquid-liquid chromatography
• Paper chromatography
• High performance liquid chromatography
Pore exchange chromatography
• Ion exchange chromatography
• High performance liquid chromatography
Permeation chromatography
• Size exclusion chromatography
Affinity chromatography
• DNA affinity chromatography
1.2 HPLC
Liquid chromatography is the most widely used of all of the analytical separation techniques. The reasons for the popularity of the method are its sensitivity, its ready adaptability to accurate quantitative determinations, its easy to automation, its suitability for separating non-volatile species or thermally fragile ones, and above all.1The first instrumental liquid chromatography was constructed by Csaba Horvath at Yale University in 1964, and was described as a high-pressure liquid chromatography (HPLC).3
High-pressure liquid chromatography (HPLC), sometimes called high-performance liquid chromatography, is a separation technique based on a solid stationary phase and a liquid mobile phase. Separations are achieved by partition, adsorption, or ion-exchange processes, depending upon the type of stationary phase used. HPLC has distinct advantages over gas chromatography for the analysis of organic compounds. Compounds to be analyzed are dissolved in a suitable solvent, and most separations take place at room temperature. Thus, most drugs, being non-volatile or thermally unstable compounds, can be chromatographed without decomposition or the necessity of making volatile derivatives.

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