Thin Layer Chromatography - Analysis

Analysis

As the chemicals being separated may be colorless, several methods exist to visualize the spots:

  • Often a small amount of a fluorescent compound, usually manganese-activated zinc silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence.
  • Iodine vapors are a general unspecific color reagent
  • Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate
    • Potassium permanganate - oxidation
    • Iodine
    • Bromine
  • In the case of lipids, the chromatogram may be transferred to a PVDF membrane and then subjected to further analysis, for example mass spectrometry, a technique known as Far-Eastern blotting.

Once visible, the Rf value, or retardation factor, of each spot can be determined by dividing the distance the product traveled by the distance the solvent front traveled using the initial spotting site as reference. These values depend on the solvent used and the type of TLC plate and are not physical constants.

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