Staining reagents

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Chapter 7

Specific Staining Reactions

An undoubted advantage of TLC is the simplicity of qualifying a separation. Coloured compounds can be seen by eye. Compounds absorbing only below 400 nm can be visualized by incorporating a fluorescent dye in the layer. Illuminating the plate with light of 256 nm will induce fluorescence. A sample zone covers the dye and inhibits fluorescence, so the zone is visible as a dark band. Illuminating the plate with light of 366 nm can identify substances with native fluorescence. Under these conditions you can observe most of the fluorescent substances by eye. If all the above-mentioned methods fail, TLC separations can be visualized without destruction by exposing the layer to iodine vapour. Some iodine crystals stored in a closed jar instantly form a violet vapour phase. Placing a plate in this jar will enrich the stationary phase with iodine. Lipophilic sample zones will enrich iodine more efficiently than clean areas of the plate. Substance zones are visualized as brown bands on a nearly white background. Removing the plate from the iodine vapour causes the iodine to evaporate. The visualization procedure also works in reverse. Iodine is removable without any reactions from the separated zones. The duration of staining is sufficient for taking a photograph. It is insufficient for densitometric measurements, but staining can be irreversibly fixed when the plate is dipped for 2 s in a 0.5% (w/v) aqueous starch solution. Figure 7.1 shows an HPTLC plate stained with iodine vapour. The sensitivity of this staining can be seen at the lower middle of the plate. A brown spot indicates fat from a thumb print because the plate was held without gloves. Water is another universal detection reagent for lipophilic compounds. The plate is dipped in water for 1 s and then wiped with a window wiper. The plate is placed on a black background. The wetted plate is translucent except where the lipophilic zones are located. Here the plate is not wetted enough to let the black colour of the background pass. As a result the plate shows white zones on a black background. Figure 7.2 shows a fungicide separation on silica gel. The lipophilic fungicides are visible as white zones on a black background. After drying, the zones can be made visible once again. The method is suitable for taking a photograph. Densitometric measurements are not possible.

B. Spangenberg et al., Quantitative Thin-Layer Chromatography, DOI 10.1007/978-3-642-10729-0_7, # Springer-Verlag Berlin Heidelberg 2011

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