Science of Chromatography

Cladosporium bio assay is another identification method specific for the detection of antifungal compounds. Anti-fungal compounds are the substances that can act against fungi. In this assay Cladosporium spp. are used because of the characteristic features of their mycelium and the spores. The mycelium and spores of Cladosporium sp. is dull black in colour. This feature detects the anti-fungal compounds in contrast.  The spores of Cladosporium sp. (Microscopic view) The mycelium of Cladosporium sp. First a chromatogram was performed with the substances that are having different anti-fungal activities. Next a suspension of conidia of Cladosporium sp. is prepared. Then this is sprayed over the TLC plate. The TLC plate is then incubated. After incubation some areas can be seen in white where as rest of the whole area is black in colour. The black colour is due to the development of conidia of Cladosporium sp. White areas are the places where anti-fungal compounds are p

Identification is usually carried out by scraping off the eluted spots on the chromatogram and eluting them in analytical columns. The identified compounds then quantified using several methods. These can be quantified by densitometer, spectrophotometer,auto-radiography and fluorescence. Densitometry : Measuring optical density in light sensitive materials is done using a densitometer. This study is known as densitometry which is a quantitative study. Light sensitive materials are photographic paper or photographic films. The result of the darkness of a developed picture is termed optical density which can be expressed as the number of the dark spots in a given area. But this is usually a relative value expressed in a scale. The densitometer can be used in spot densitometry, line densitometry and bi-dimensional densitometry. Fluorescence : Fluorescence is the emission of light by compounds which have absorbed light or other electromagnetic radiation already. Usually

Thin layer chromatography (TLC) plate is prepared by application of a uniform layer of adsorbent on to a glass or an Aluminum plate. The application of the adsorbent can be done by pouring the slurry, spraying the suspension and use of commercial applicators. The slurry is made by dissolving SiO 2 in water. Also there are readymade TLC plates available in shops. Then this plate should be heated at 100 0 C -105 0 C for 30 minutes in an oven. This is to activate the plate and this activation should be done before the sample is loaded. There after the sample is loaded. When the sample is loading we must be sure to spot the sample several times in order to obtain a concentrated spot. But the diameter of the spot should not be exceeded 2mm. If the spot is large, then during the separation the lines of the compound will be not limited to the plate (B). And also if the spot is big and dilute then a peak of the line is difficult to observe. Then the plate is inserted into th

Thin layer chromatography is another type of adsorption chromatography. Thus the basis for separation is the polarity. Polarity of the compounds in the test sample is compared with the polar stationary phase. The polar stationary phase is due to the presence of SiO 2 . When consider the polarity of mobile phase, it should always be relatively less polar than the stationary phase. Thus when choosing an organic compound as a mobile phase we must be highly concern of the polarity of the organic solvent. The glass solid support or Al solid support is applied with thin slurry of the SiO 2 and the test sample is spotted at the base line. This spot of the test sample should be a very concentrated but a minute one! With the rise of the mobile phase the spotted test sample is move up wards. Polar substances of the test sample are interacted with the stationary phase and travel slowly along the solid support. Less polar substances travel faster with the solvent.