Development of Thin Layers - Thin Layer Chromatography (TLC)

DEVELOPMENT OF THIN LAYERS

The spotted TLC plates, after evaporation of the sample solvent, is placed in a closed chamber saturated with vapours of the developing solvent(s). One end of the plate is then wetted with the developer by means of either ‘ascending-technique or the ‘descending-technique’ as shown in Figure 28.2 (a), (b). After the devel-oper has traversed one-half to two-thirds the total length of the TLC plate, the latter is removed from the chamber, air-dried and the positions of the components are located by any of several methods.

There are three major factors which essentially govern the ‘development of thin-layers’, namely :

(i) Equilibration of the chamber (or chamber-saturation),

(ii) Protection against oxidation (temperature and light), and

(ii) Visualization.

1. Equilibration of the Chamber

The equilibration of the chamber or chamber-saturation is a vital factor to obtain reproducible Rf values. It may be achieved by allowing the solvent system to remain in the chamber for at least 1 to 2 hours so that the vapours of the solvent(s) would pre-saturate the latter adequately. This is done to obtain distinct separation of constituents, uniform solvent from and prevent evaporation of the solvent on TLC-plates.

2. Protection against Oxidation

Both temperature and light augments oxidation and, therefore, ideally the following experimental parameters must be observed to obtain the best development of thin-layers, viz.,

Temperature : 18-23°C, and

Light                : Diffused daylight both natural and artificial,

However, direct sunlight (UV) or drought may give rise to ‘oblique formation’ of the solvent front.

3. Visualization

As a result of both intensive as well as extensive research a number of organic and inorganic substances have been identified that positively demonstrate an ‘improved visualization’. Such substances are termed collectively as ‘fluorescent indicators’.

Examples : Barium diphenylamine sulphonate ; 2,7-dichlorofluorescein ; Fluorescein (0.2% w/v in Ethanol) ; Morin (0.1% w/v in Ethanol) ; Sodium fluorescinate (0.4% w/v in water) ; Rhodamine B ; Zinc Silicate ; Calcium silicate ; Methylumbelliferone (or 7-hydroxy-4-methyl coumarin).