Revelation of spots on paper and thin-layer chromatograms

Revelation of spots on paper and thin-layer chromatograms 

Substances separated on paper and thin-layer chromatograms are revealed or detected by derivatization to coloured or fluorescent products, and these are interpreted visually, evaluated semi-quantitatively with reference to standards run under identical conditions, or quantitated by elution of the coloured or fluorescent spots and instrumental determination by colorimetry or fluorimetry respectively. In paper chromatography the spots can be cut out with scissors or removed with a paper punch thin layers are usually scraped off the support and analysed in some appropriate way. A neat application is to analyse TLC spots directly by FAB MS [8], and this potentially provides identification as well as quantitation. It is one of the advantages of paper chromatographic and TLC methods that several samples and standards can be run under identical conditions, but the accuracy and precision of quantitation by these means are usually not nearly as good as with liquid or gas-liquid chromatographic separations. 

Substances on paper chromatograms are generally revealed by spraying or dipping. It is important to make sure that the reagents used are dissolved in a solvent which will not smear or diffuse the developed spots and 

Many commercial thin-layer media incorporate a fluorescent indicator, and under ultraviolet light the separated spots may show up dark against the brightly fluorescent background, but a host of substances will not show up under ultraviolet light on these fluorescent layers. 

There must be hundreds of visualization recipes, some of them highly ingenious, particularly where sequences of several colorimetric or fluorimetric methods have been elaborated to provide highly specific identification of certain compounds. These visualization methods have been gathered into various handbooks on paper and thin-layer chromatography [4, 11-14], and the wealth of applications they contain is far more abundant than can be included here. 

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