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Identification and Quantification in Thin Layer Chromatography

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 the wave length of emitted radiation is longer than the absorbed radiation. Which means emitted light is higher in energy than the absorbed light.   Many analytical methods involve the use of a fluorometer. In TLC, if compounds are fluorescent then TLC plates can be visualized.



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