Medical brain notes 14

  INTRODUCTION Thin-layer chromatography  (TLC) is an altogether new, versatile and specialized laboratory technique   that was evolved in early Fifties, and since then has become an indispensable means of separation for analysts and researchers round the globe. It can be employed conveniently both for organic and inorganic substances, either derived from natural sources or synthesized in the laboratories, on quantities ranging from the nanogram to microgram levels.   Kirchner in 1950 was the first who used adsorption chromatography on impregnated glass-plate coated with silicic acid or alumina. It may be emphasized, however, that  Egon Stahl’s fundamental work  stands as a landmark in the world-wide acceptance of this new technique in the laboratory. Later on, Stahl in 1958, introduced a standard equipment for preparing uniform thin-layers of known thickness, which eventually led to the ultimate acceptance of this new technique as an additional modern tool for analytical chemistry.  

  THEORY The adsorbent used in TLC is a thin, uniform layer (normally 0.24 mm thick) of a dry, finely powered material applied to an appropriate support, such as a glass plate or an aluminium sheet or a plastic foil. Subsequently, the mobile phase is permitted to move across the surface of the plate (usually by capillary action) and the chromatographic phenomenon may solely depend upon adsorption, partition, or a combination of both, depending on the adsorbent, its treatment, and the nature of the solvents employed. During the chromatographic separation procedure the TLC-plate is placed in a chromatographic chamber, mostly made up of glass to enable clear observation of the movement of the mobile phase up the plate, that is pre-saturated with the solvent vapour. The inert solid supports invariably employed are, namely : alumina, silica gel, kieselguhr and cellulose, to these may be added appropriate substances, for instance : calcium sulphate (gypsum) so as to provide adequate adhesion

  PREPARATION OF THIN LAYERS ON PLATES   The paramount importance with regard to the preparation of thin layer is that it must be uniform and consistent throughout. Various means have been put forward to apply thin layers of powdered or their suspen-sions or their slurries to the carrier plates with a view to achieve an uniform layer throughout the length of the plates. These are namely :   ( a )  Pouring of Layers :   In order to obtain layers of equal thickness, a measured amount of the suspen-sion or slurry is placed on a given-size plate that is rested on an absolutely labelled surface. The plate is subsequently tipped backward and forward to permit the slurry (or suspension) to spread uniformly on the surface of the plate.   ( b )  Dipping :   Peifer* in 1962, was pioneer in introducing this technique, whereby two plates at a time back-to-back are dipped together in a slurry of the adsorbent in either chloroform or chloroform-methanol. However, this particular methods is not much

  CHOICE OF ADSORBENTS   The choice of proper adsorbent in TLC plays a vital role in the separation of components either belong-ing to natural origin or to purely synthetic origin. It is chiefly based on certain crucial informations like :   ( i ) Solubility of the substance  e.g. , hydrophilic and lipophilic,   ( ii ) Nature of the compound  i.e. , whether it is acidic/basic/neutral/amphoteric   ( iii ) Reactivity of compound with either the solvent or the adsorbent, and   ( iv ) Chemical reactivity of compounds with the binders.   In actual practice, the adsorbents are of two types : firstly the  inorganic , and secondly, the  organic  adsorbents. A host of substances from each type are used in TLC and these shall be discussed briefly as below :   1. Inorganic adsorbents   These are namely :   ( i )  Aluminium oxide- (Al 2 O 3 ) :  The alkali (Na 2 CO 3  ; NaHCO 3 ) present in alumina very often gives rise to secondary reactions that may be eliminated by washing with dilute mineral a