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I am studying chemistry, and I am learning about chromatography.

But I am confused about how chromatography is used to ‘separate and purify’ substances.

I have learnt about chromatography paper, which is used to check if a substance if pure or not. Spots of the substances to be tested are place on the chromatography paper, which is then dipped in solvent. If there are several components, the spot separates into several spots (1 per component) as they travel up the paper.

Chromatography apparatus

Is it possible for separated and purified components obtained from the separate spots on the chromatogram, as usable substances, or do chromatographs only show the purity of substances?

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    $\begingroup$ Have you tried a basic searching before asking ? See e.g en.wikipedia.org/wiki/Column_chromatography $\endgroup$ – Poutnik Jan 5 at 16:40
  • $\begingroup$ If HPLC is available, then: 1. You first take small aliquots of the sample, inject them and try out different methods. This allows choosing the best method and finding at which Retention Time you can expect the analyte to elute 2. Now inject the rest of the sample and direct the chromatography output to different vials depending on previously known Retention Time 3. Now you can dry down that vial which gives you the purified substance 4. You may want to dilute it back to the right concentration and run another LCMS for Quality Control. $\endgroup$ – Stanislav Bashkyrtsev Jan 11 at 5:53
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Generally, all chromatography is done on silica gel. It's the exact same procedure for TLC (thin layer chromatography), except that instead of paper you have a thin layer of glass covered with silica. What can be done in that case is scraping the spots, then extracting the product with organic solvents and filtration. There exists plates made for this, which are thicker than just for analysis.

Of course this is not viable for large amounts of products. In that case, we use a column of silica (see the image below, taken from https://bitesizebio.com/29947/basics-chromatography-column/). https://bitesizebio.com/29947/basics-chromatography-column/

This time, gravity makes the solvent go through the column and come out at the other end. Compressed air is often used to make this process go much faster.

You will however think "how do you reveal the products IN the column?" As the products aren't always colored, you can't really tell when what you want is coming out. Therefore, everything is collected in tubes of the appropriate size and revealed afterwards. If everything goes right, there is no overlap between your products and you get everything out of the column. If there is an overlap, you have to either do the column again for the mixed fractions or just discard those and keep the tubes containing only the pure product you want.

This technique is used pretty much all the time, as it can separate anything (as long as it doesn't degrade on silica) and can be adjusted by changing which solvent or mix of solvents you use.

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  • $\begingroup$ In reality the outflow is monitored in almost real time by quickly sampling the portion and checking if it contains eluate(s), and eventually running a quick TLC of it. $\endgroup$ – Alchimista Jan 6 at 7:24
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    $\begingroup$ “All chromatography is done on silica gel” is not remotely true. $\endgroup$ – Andrew Jan 6 at 19:28
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There are many different ways of using chromatography. You may use it as an analytical tool, like in your figure. But you may also use it for preparatory goals. For example if you have a liquid containing some impurity that you know is adsorbed on silica or alumina, you may use a long and broad glass tube, put it in a vertical position and fill it with alumina or silica powder. Then you pour your impure liquid into the tube. The liquid will slowly get through the powder and get out in a pure state. The impurity will be fixed in the alumina or silica powder

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