Thin layer chromatograms, quantification

Quantification of thin layer chromatograms can be performed indirectly after scraping off the separated zones of samples and standards, and elution of the substances from the layer material with an appropriate solvent. The volumes of the eluates are adjusted and the solutions analyzed by use of a spectrometric method, GC, or HPLC. Scraping and elution are usually performed manually. Although direct quantification has become increasingly important, indirect analysis is still widely used (e.g., for assay of some drugs according to the USP). 

Campbell, A. Chejlava, M. Sherma, J. Use of a modified flatbed scanner for documentation and quantification of thin layer chromatograms detected by fluorescence quenching. J. Planar Chromatogr.-Mod. TLC 2001, 16 (3), 244-246. 

Methods for the quantitative evaluation of thin-layer chromatograms can be divided into two categories. In the first, solutes are assayed directly on the layer, either by visual comparison, area measurement, or densitometry. In the second, solutes are eluted from the sorbent before being examined further. Quantification of radioactive zones is not considered here but is discussed in Chapter 13. 

The various peptides and proteins have been located on the thin layer chromatograms by using ninhydrin, fluorescamine, ortho- phthaldialdehyde, iodine vapors or UV the quantitation has been performed by densitomelric scanning or by spectrophotometry after eluting the peptides immuno-chromatography using monoclonal antibodies has also been used for quantification. 

Bottleneck 2. From the results discussed in this section it clearly comes out that densitometric detection applied to the thin layer chiral separations has to be performed in a very cautious and also very conscious manner, that is, anticipating the possibility of quite significant deviations of the chiral analytes migration tracks from the straight-line direction. In this sense, the present section contains a troubleshooting advice addressed to all those, who use TLC with densitometric detection for chiral separations and — what is even more important — for quantification of the analyzed mixtures composition. The recommended troubleshooting solution is to scan relatively broad bands on the respective chromatograms instead... 

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