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Define about the Column chromatography?
In column chromatography, a tube is filled with the material constituting the stationary phase, plus a solvent (mobile phase). The general adsorbent (stationary phase) used are alumina, silica gel, activated carbon, calcium phosphate and hydroxyapatite. The substances to be separated are dissolved in the smallest possible volume of a suitable solvent and applied on the top of the stationary phase and allowed to enter the column. The chromatogram is then developed by flowing a solvent (the mobile phase) through the column. As different substances move through the column, they separate and appear in the effluent when particular volumes of liquid have passed through the column. The liquid leaving the column (the eluent) is usually collected as discrete fractions, using an automatic collector.
The separated components are then identified by testing aliquots of each fraction by one or a combination of the following methods viz. colorimetry, UV absorption, fluorimetry, scintillation counting, refractive index, spectral diode array system, electrochemical detection, radioimmunoassay and enzyme immunoassay. When automatic fraction collector is used the recorder automatically draws each peak and the area of each peak is proportional to the amount of sample component present in it. The simplest form of column chromatography is adsorption chromatography. Separation of components by this method depends upon differences both in their degree of adsorption by the adsorbent and solubility in the solvent used for separation. These physiochemical factors are governed by the molecular structure of the compound.
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