Surface-modified phases (reversed phases, RP):<\/strong> These are phases in which the polar groups of the silica gel have been chemically modified by the attachment of non-polar hydrocarbon chains (e.g. C8, C18). In this system, the stationary phase is non-polar and the mobile phase is polar (e.g. mixtures of water with methanol or acetonitrile). This mechanism is called reversed-phase chromatography (RP-TLC).<\/li>\n<\/ul>\n\n\n\nBelow are examples of the use of different stationary phases:<\/p>\n\n\n\nAdsorbent<\/th> Separation mechanism<\/th> Example of analytes<\/th><\/tr><\/thead> Silica gel<\/td> adsorption, partitioning<\/td> almost all<\/td><\/tr> Aluminium oxide<\/td> adsorption, partitioning<\/td> alkali and steroids<\/td><\/tr> Siliceous soil<\/td> partiotioning<\/td> sugars, higher fatty acids<\/td><\/tr> Magnesium phosphate, magnesium silicate, calcium hydroxide, calcium phosphate<\/td> adsorption<\/td> karetonoids and tocopherols<\/td><\/tr> Coal<\/td> adsorption<\/td> non-polar compounds<\/td><\/tr> Ion exchangers<\/td> exchange<\/td> nucleic acids<\/td><\/tr><\/tbody><\/table><\/figure>\n\n\n\nHow to select the mobile phase?<\/h2>\n\n\n\n The selection of the mobile phase (eluent) is an optimisation process that aims to achieve the best possible separation (differences in migration velocity) of the analytes. The key principles are:<\/p>\n\n\n\n
\nThe \u201clike dissolves like\u201d rule<\/strong> is reversed here: in order for a substance to move across a plate, the mobile phase must have the ability to \u201cwash\u201d it from the stationary phase.<\/li>\n\n\n\nPolarity of the eluent:<\/strong> The elution power of a solvent (its ability to move analytes up the plate) depends on its polarity.\n\nIn the case of normal phases (e.g. silica gel), the more polar the eluent, the more strongly it interacts with the stationary phase, competing with the analytes for active sites and thus accelerating their migration.<\/li>\n\n\n\n In the case of reversed phases, the less polar the eluent, the greater its elution force.<\/li>\n<\/ul>\n<\/li>\n\n\n\n Eluotropic series:<\/strong> In thin-layer chromatography, it is not possible to perform gradient separation (such as in HPLC). Instead, a so-called eluotropic series can be used. In short, one uses successive solvents of increasing elution strength. Example series (from weakest to strongest eluent): hexane < toluene < chloroform < ethyl acetate < acetone < ethanol < methanol.<\/li>\n\n\n\nSolvent mixtures:<\/strong> A single solvent is rarely used. Most often mixtures are used, e.g. hexane with ethyl acetate. By varying the proportions of the components, the polarity of the eluent can be precisely 'tuned’ and optimal Rf values can be obtained (see below).<\/li>\n<\/ol>\n\n\n\nTLC chromatogram staining and detection<\/h2>\n\n\n\n If the separated substances are colourful, they can be observed with the bare eye. However, they are usually colourless and require special detection methods:<\/p>\n\n\n\n
\nUV light:<\/strong> Many TLC plates contain a fluorescent indicator that emits green light when exposed to UV light at 254 nm. Compounds that absorb UV light quench this fluorescence and appear as dark spots. Other compounds may themselves fluoresce.<\/li>\n\n\n\nIodine vapour:<\/strong> The plate is placed in a chamber saturated with iodine vapour. Iodine is adsorbed onto the surface of the analytes (especially organic compounds), leading to the appearance of yellow or brown spots. This method is versatile and usually reversible.<\/li>\n\n\n\nStaining reagents (sprays):<\/strong> These are chemical solutions with which the plate is sprayed. They react with specific functional groups of analytes to give coloured products. Here are examples of some staining reagents\n\nNinhydrin: for detection of amino acids (gives purple spots).<\/li>\n\n\n\n Potassium permanganate (KMnO4)<\/strong>: for the detection of readily oxidisable compounds, e.g. unsaturated compounds (yellow spots appear on a violet background).<\/li>\n\n\n\nVanillin solution in sulphuric acid: universal stain for many organic compounds, gives a wide range of colours when the plate is heated.<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n\n\n\nCalculations<\/h2>\n\n\n\n Staining the chromatogram, making the spots visible, is only the beginning of the analysis of the results. Each spot is one of the separated substances. They still need to be identified. A basic parameter that helps to identify substances is the retardation factor (Rf)<\/p>\n\n\n\n
![\"Thin](\"https:\/\/bioeducator.eu\/wp-content\/uploads\/2025\/07\/TLC_black_ink_cropped.jpg\")
![\"Thin](\"https:\/\/bioeducator.eu\/wp-content\/uploads\/2025\/07\/TLC-1024x465.png\")
![\"Rf](\"https:\/\/bioeducator.eu\/wp-content\/uploads\/2025\/07\/Rf-english-300x278.png\")