6 Use of Phosphorescent/Fluorescent Indicators

To aid absorbance many commercial pre-coated TLC layers contain an inorganic phosphorescent indicator, shown on the label by the designation F ₂₅₄ or UV ₂₅₄. Most indicators exhibit a bright green, yellow or blue phosphorescence when excited by UV light at 254 nm. Indicators that have been used are uranyl acetate (yellow-green), manganese zinc silicate, zinc cadmium sulphide, zinc silicate (green), alkaline earth metal tungstates, and tin strontium phosphate (blue). Detection by absorbance in these cases relies on the phosphorescence being quenched by the sample components. In most cases pink or violet spots/bands are observed for quenching on the green phosphorescent background, whereas grey-black spots/ bands are usually seen on the blue background. In most applications the inorganic indicators are quite stable with little or no elution from developing solvents, and they remain unaffected by most dyeing reagents and the temperatures used to effect reactions. Organic fluorescent indicators can also be used. These are coded F ₃₆₆ or UV ₃₆₆ by the manufacturer. Substances used include optical brighteners, hydroxypyrene sulfonates, fluorescein and rhodamine dyes. As indicated by the coding used, these organic indicators fluoresce in long wave UV light (at 366 nm). Some pre-coated plates are manufactured with both types of indicator present to give possible quenching by sample components at both wavelengths. Sometimes the designation F _254s is used in the description of some TLC and HPTLC plates. The "s" simply indicates that the fluorescent indicator is acid resistant,...