Chromatography Forum

Hi all,

I have been using aluminium oxide (alumina) chromatography to separate/purify components from a plant extract for a few years with reasonable success. The alumina is active basic (Brockmann activity I).

Recently the chromatography has resulted in the compound of interest (CoI) to elute with the more polar compunds in the extract as if it wasn't binding to the alumina at all. In the past the CoI would bind to the alumina and elute after the polar impurities had eluted.

The mobile phase begins with a 50:50 mixture of acetone and heptane, and the acetone is ramped up slowly to 100%. This has not changed. One clue is that I am getting a red band on a silica TLC plate (I spot each fraction on a TLC plate to identify the CoI after chromatography) with low Rf that I believe to be chlorophyll on the separations that fail. These red bands appear in similar fractions to the CoI.

Is it possible that chlorophyll deactivates or binds strongly to the alumina, thereby preventing adsorption of the CoI?

Any help would be appreciated.

Thanks,
mlh_pep.

Hi,

I will admit that I am not really a chromatographer, but I do use an LC system in my work and I have interest in adsorption processes/mechanisms at liquid solid boundaries, especially those of environmental relevance. I am only vaguely familiar with the structures of plant components such as yours, but it is well established that aluminum oxides are active towards the adsorption of a range of species in solution. In particular, there are Al-OH groups at the surface that are believed to be able to exchange with carboxylate groups to give Al-O2CR groups, i.e. molecules attached via actual covalent linages.

However, I am not sure what are the modifications of Al oxide are that you are working with... so you may discount this offhand..

Regards,

David

Just to clarify: more polar constituents will elute later on an alumina (normal phase) column. Chlorophyll is non-polar, which is why it elutes with low Rf in your system. I doubt that chlorophyll is binding stongly to the alumina. What might be happening is that the chlorophyll could be binding to your compound making it more non-polar so that it elutes earlier.

Thanks dkreller and sassman. Interesting points.

Sassman, I got my polarities mixed up. You are correct in pointing out that more polar compounds will elute later than less polar on alumina. If chlorophyll is binding to my CoI, thereby making it effectively less polar, then it could well explain what I am seeing. I'll need to have a bit more of a think about it, but thanks for the suggestion. You've put my thinking on a different, and probably more relevant, path now.

mlh_pep.