Clever Chromatography

What if the substances we’re interested in are colourless? There aren’t any spots to see!

There are two simple ways of getting around this problem.

Using fluorescence

If the stationary phase ( the solvent part) has a substance added to it which fluoresces or glows when exposed to UV light, the glow is masked at the position where the spots are on the final chromatogram – even if those spots are invisible to the eye.

chromo

That means that if you shine UV light on the paper, it will all glow apart from where the spots are. The spots show up as darker patches, like this.

While the UV is still shining on the plate, you obviously have to mark the positions of the spots by drawing a pencil circle around them. As soon as you switch off the UV source, the spots will disappear again.

Showing the spots up chemically

In some cases, it’s possible to make the spots visible by reacting them with something which makes a coloured product. A good example of this is in chromatograms produced from amino acid mixtures. Amino acids are the building blocks of proteins.

The chromatogram is allowed to dry and is then sprayed with a solution of ninhydrin. chromo2Ninhydrin reacts with amino acids to give coloured compounds, mainly brown or purple. Have a look.

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 vapour 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 we’re interested in may show up as brownish spots.

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