Chemicals:
| Test solution: Dye mixture (Butter Yellow, Sudan Blue II,
Sudan Red G and Indophenol in toluene.
| | Reference solutions: Sudan Red G and Sudan Blue II each dissolved in toluene.
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| Developing solvent: toluene |
Glass wares and materials:
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| developing chamber (jam glass with a screw cover h = 11 cm d = 5 cm )
| | DC plate POLYGRAM® ALOX N/UV254 (Macherey Nagel)
| | glass capillaries (1 µL) |
Hazards and safety precautions:
  | Toluene is toxic by inhalation, ingestion or by absorption through skin. Serious irritant. Experimental teratogen. |
Safety goggles and gloves required. The experiment should be performed in a portable laboratory fume hood giving all-round visibility!!
Experimental procedure:
Using a soft pencil, a line is drawn approximately 1 cm from the bottom of the plate. The spotting points are numbered (1,2,3). At the spotting points 1 and 3 the reference solutions are applied onto the plate, at the spotting point 2 the dye mixture. Using capillaries approx 1 µL of the dye solutions are applied to the TLC plate. The capillaries fill themselves quickly when dipped into organic sample solutions. Before emptying the submerged end of the capillary is rolled horizontally on filter paper. The clean upper end of the capillary is placed on the layer vertically and carefully, vertically so that the capillary empties itself and carefully to avoid damage to the layer. Easy application of samples is allowed with a spotting guide.
When the solvent is completely evaporated (approx. 10 min) from the plate, the loaded TLC plate is carefully placed in the TLC chamber with the sample line toward the bottom. The plate whose top is leaned against the jar wall should sit on the bottom of the chamber and be in contact with the developing solvent (solvent surface must be below the extract line). The TLC chamber is covered. The TLC plate is allowed to remain undisturbed. When the solvent front has reached three quarters of the length of the plate, the plate is removed from the developing chamber and the position of the solvent front is immediately marked. The solvent on the plate is allowed to evaporate.
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Videoclip 
