Thin Layer Chromatography (TLC): Theory

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 to the solid support, example : silica gel-G (G-stands for gypsum).

The prepared layer may be impregnated with suitable materials to achieve specific purpose, namely :

(i) Buffering materials : To afford acidic, basic or neutral layers,

(ii) Silver nitrate : To modify its characteristics e.g., for separation of methyl esters of fatty acids, and

(iii) Ion-exchange materials : To modify its properties, e.g., admixture of cellulose with ion-exchange resins used for the separation of nucleic acids and their respective derivatives.

Therefore, the application of skill and wisdom may give rise to a fairly wide spectrum of possible layers, employed in conjunction with a vast combination of solvent systems permits and affords an almost infinite variation of separating power that really makes TLC such a versatile and useful technique in the domain of pharmaceutical analysis.

1. VERSATILITY OF TLC OVER PAPER AND COLUMN CHROMATOGRAPHY

In general, TLC essentially not only amalgamates the meritorious plus points of both paper and column chromatography but also it is distinctly superior and more versatile to either of the two methods. However, the versatility of TLC over paper and column chromatography are quite evident from the following points, namely :

(i) Simple equipments : TLC mostly requires very simple equipments, such as : micro-slides ; specimen jars with lid ; glass-sprayers ; strips of glass sheet ; small chromatank etc.

(ii) Short development time : In TLC, the separation is very rapid i.e., the development time is of short duration (say 1 hour) for reasonably good separation on inorganic adsorbent layers. Hence, it has a positive edge over paper and column chromatography which normally takes several hours or days.

(iii) Wide choice of stationary phase : TLC may be used for adsorption, partition (including reversed phase) or ion-exchange chromatography,

(iv) Quick recovery of separated constituents : TLC permits the possibility of removal of the adsorbent coating on the plates by scraping with a spatula. In other words, a spot or a zone can be removed quantitatively, and the separated constituent dissolved in an appropriate solvent is estimated either by suitable spectrophotometric or colorimetric analysis.

(v) Separation effects : The separation effects obtained by TLC are more distinctive and superior than those of paper chromatography,

(vi) Easy visualization of separated components : Detection of fluorescence components when exposed to UV light is much easier than on paper by virture of the fact that inorganic material (i.e., adsorbent) has intrinsic fluorescence,

(vii) Detection Limit : TLC affords extremely sharp delineated spots and offer lower detection limit i.e., one decimal power less than that in paper chromatography,

(viii) Variable thickness of think layers : The method employed in TLC may be further extended to preparative separations by using thicker layers and also to meet separations by column chromatog-raphy,

(ix) Chemically inert stationary phase : Use of inorganic adsorbents e.g., alumina and silica, in TLC allows the application of corrosive sprays to detect fractionated substances, for instance : carbohy-drates by 70% conc. H₂SO₄, and

(x) Trace analysis : TLC method is suitable as micromethod in trace analysis.