Download: Cited By: Authors: Archana A. In some cases, it may be possible to make the spots visible by reacting them with something which produces a coloured product. A good example of this is in chromatograms produced from amino acid mixtures. The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple. In another method, the chromatogram is again allowed to dry and then placed in an enclosed container such as another beaker covered with a watch glass along with a few iodine crystals.
The iodine vapor in the container may either react with the spots on the chromatogram, or simply stick more to the spots than to the rest of the plate. Either way, the substances you are interested in may show up as brownish spots. Suppose you had a mixture of amino acids and wanted to find out which particular amino acids the mixture contained. For simplicity we'll assume that you know the mixture can only possibly contain five of the common amino acids.
A small drop of the mixture is placed on the base line of the thin layer plate, and similar small spots of the known amino acids are placed alongside it. The plate is then stood in a suitable solvent and left to develop as before. In the diagram, the mixture is M, and the known amino acids are labelled 1 to 5. The left-hand diagram shows the plate after the solvent front has almost reached the top.
The spots are still invisible. The second diagram shows what it might look like after spraying with ninhydrin. There is no need to measure the R f values because you can easily compare the spots in the mixture with those of the known amino acids - both from their positions and their colours. In this example, the mixture contains the amino acids labelled as 1, 4 and 5.
And what if the mixture contained amino acids other than the ones we have used for comparison? There would be spots in the mixture which didn't match those from the known amino acids. You would have to re-run the experiment using other amino acids for comparison.
The stationary phase - silica gel. Silica gel is a form of silicon dioxide silica. The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups. The diagram shows a small part of the silica surface. Confinement of a TLC plate in a chamber which has its headspace the air in the chamber saturated with solvent vapor allows for elution of a sample by capillary action.
The solvent simply rises up the slide and brings the analyte with it. Solvents are not strictly one and only one compound. Different solvents can be mixed in varying ratios to achieve a good separation.
Determining the optimum solvent mixture for your TLC experiment can be challenging, as there are no steadfast rules governing this procedure. It is almost entirely a matter of building experience through trial and error.
How come we can't see anything? That's because usually you need something like a UV lamp shining on this, so that you can visualize something. So let's say we take our UV lamp and shine it on here. Compounds that are aromatic will usually show up and fluoresce. And let's say that we had these two dots.
So what does that tell us? That tells us that whatever was in our reaction flask was a two-component mixture and that there's at least two compounds in there. However, you would have seen more dots if there were three compounds or four compounds or even more.
TLC plates can get really messy when you're doing research. But what can we tell about these two compounds? So TLC is a pretty qualitative method. It'll tell us whether things are more polar or less polar, so what we have to keep in mind is that the stationary phase, where the silica gel-- silica gel is very, very polar. So you can see this one didn't move to far-- it means it must have been really attracted to the silica gel-- but this one moved a lot more, so this is less polar and more attracted to the mobile phase.
So we still don't really know what compounds these are exactly. But let's say that you knew that inside the flask you had naphthalene and benzoic acid.
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