After drying of the developed chromatograph, it is very essential to locate the separated substances. If the substances are coloured, no difficulty arises but the problem stands with the colourless substances. There are several methods which have been described to locate the spots, all of which can be grouped into 1 ) Chemical and (2) Physical methods.
The locating reagents are applied by the following methods:
(1) Dipping the paper into the reagent solution. (2) Spraying the solution on the paper
The DIPPING TECHNIQUE
In this method it is essential to choose a solvent in which the substances are insoluble. Dipping is done in an inert material tray of smaller size. The volatile solvents are most suitable because they are rapidly evaporated from the chromatogram. In some cases e.g. for location of Ag, Pb, and Hg 0.25 MnKCro4 solution is used in which the chromatogram is dipped. The paper is washed continuously under a stream of water so that excess of locating reagent is washed away and all that left is the coloured spots of the substances. The most widely used reagent for detecting amino acids (the building, blocks of proteins) is Ninhydrin (0.2-0.3%) Solution experience has shown that for dipping acetone is better than other solvents for ninhydrin. Since ,the colour is produced more rapidly and is more intense. paper is removed and is heated at 10 5 C for 5-6 minutes to allow the lilac spots are visible. The dipping technique. cannot be used if either the compounds on the chromatogram or their coloured reaction products are soluble in the solvent used for the locating reagent. therefore, in that case spraying is recommended.
THE SPRAYING TECHNIQUE
The solution of the locating reagent is sprayed uniformly over the surface of the chromatogram with the help of a glass atomizer or scent spray. The Chromatogram are hanged on suitable racks and fixed in the position with the help of stainless steel clips. The atomizer held in the position normally at a distance of l2″- 15″ from the paper and is moved slowly from top to bottom in a left to right direction .lt is to be noted that much of the spray is not done on the paper . This will result in the diffusion of the spots.
In such cases where the compounds are invisible in ordinary light. UV lamp can be used to locate their position. The other method is the use of radioactivity in which a number of labelled radioactive compounds are available which can be detected by means of Goiger Muller counter.
UV DETECTION A large number of unsaturated organic compounds give fluorescence .They have the property of absorbing ultraviolet light or violet light (invisible) of short wave length and emitting light of longer visible wave length. These compounds. although invisible on chromatograms in ordinary light can readily be detected under an ultraviolet lamp. The wavelength of light emitted, and hence the colour band is characteristic of the compounds and is therefore useful for the purpose of identification. Numerous fluorescent compounds are present in most plant tissues.
The sample is labelled with certain radioactive isotopes. These can be detected on chromatograms by means of a special counter.