7.3.2. Thin Layer (TLC)

Thin layer chromatography (TLC) is one of the oldest and most commonly used methods in the molecular analysis of biopigments, including those derived from fungi. This procedure involves chromatography in a stationary phase in the form of a thin layer, usually on aluminum, glass, and also plastic surfaces, where a standard solution is added and expressed as a narrow band on the thin layer of adsorbent as an indicator that it has been distributed evenly on the support/surface. This technique consists of chromatography carried out in a stationary phase in the form of a thin layer, commonly on aluminum, glass, and even plastic surfaces and with solvents as mobile phase. The separated molecules are detected after the evaporation of the solvent and by employing physical methods or chemical staining reagents [46,48,72,73].

Thin layer chromatography allows for the manipulation of extracts of different types, whether crude or pure; additionally, the equipment used is simple, inexpensive, and effective. And also, this method can be used quantitatively, where the components are isolated on the TLC tray, to be subsequently removed with an appropriate solvent, such as ethanol or acetone, and then lead to the application of a procedure using a chromogen, and thus be measured by spectrophotometry at a determined wavelength [74,75]. There are other ways for TLC quantification and detection, such as densitometry under the appropriate conditions, which is commonly used for the study of pigments, mostly anthocyanins and vitamin A precursors [76]. Another simple example is its application to the complete isolation of pigments, most notably those derived from organisms such as *Monascus*, which do not contain only one kind of pigment [77]. Finally, TLC has been used in conjunction with other approaches to increase the belief in carotenoid identifications. For example, Wang et al. [13] used both column chromatography and TLC to investigate the pigments of aeciospores of *Cronartium fusiforme* fungi [13].
