Chemicals:
| Test solution: a mixture of 7 dyes dissolved in water: Erythrosin, Brillant Black
BN, Fast Red E, Naphthol Red S, Yellow Orange S, Ponceau 4R, Tartrazine. |
| Reference solutions: Yellow Orange S and Brillant Black, each dissolved in water.
| Developing solvent: 2.5 % sodium citrate solution, ammonia 25 %, 2-propanol
(20 : 5 : 3)
The developing solvent must be freshly prepared. |
Apparatus and materials:
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| developing chamber (jam glass with a screw cover h = 11 cm, d = 5 cm)
| | Fertigfolie POLYGRAM® CEL 300 plate (Macherey Nagel)
| | glass capillaries (1 µL) |
Hazards and safety precautions:
  |
Concentrated ammonia solution is extremely damaging to eyes. Even contact with dilute ammonia solution can lead to serious
eye damage Harmful if swallowed or inhaled and in contact with skin. |
 |
2-Propanol is highly flammable. |
Safety goggles and protective gloves required. The developing solvent should be prepared in a laboratory fume hood!
Experimental procedure:
Using a soft pencil, a line is drawn approximately 1,5 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 0.25 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 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.
Results:
The dye mixture is separated into individual components. The progress of their separation may be followed by observing the
movement of colored spots. The use of referenve solutions allows to identify Yellow Orange S and Brillant Black in the mixture.
 Video clip (Download RealPlayer .rm file)
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Background:
Qualitative analysis of separated components in TLC is based on a comparison of rates of migration. The retention factor,
Rf value, is used to characterize and compare components of various samples.
The Rf value is defined as follows:
In order to get reproducible Rf vakues the atmosphere in the developing chamber must be saturated with the solvent. The
composition of the mobile phase and the temperature must remain constant.
References:
Demonstration Experiment on Video
Separation of a Lipophilic Dye Mixture by Thin Layer Chromatography (TLC)
Demonstration Experiment on Video
Separation of Plant Pigments by Thin Layer Chromatography (TLC)
Demonstration Experiment on Video
Separation of Plant Pigments by Column Chromatography (CC)
Index of Lecture Experiments
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