Chromatography Forum

Dear all,

I am relatively new to HPLC (my background is as synthetic chemist) but I am using it now daily for my current project. I am working on the separation and derivatization of sulfated oligosaccharides (di-, tetra- and hexasaccharides) from glycosaminoglycans. Reading this forum and the litterature, it seems that HILIC has become increasingly popular recently. I was just wondering whether HILIC has ever been used for the separation of sulfated oligosaccharides. If that is not the case, do you think it could be a viable and maybe more practical (for LC-MS applications) alternative to SAX-HPLC, which is currently the most used technique for this kind of molecules?
Many thanks for your suggestions

Emiliano Gemma

It definitely is a good option, and superior to ion-exchange. The tool to use is a silica column for HILIC and a mobile phase with a large excess of acetonitrile. pH control is possible, but probably not needed for the type of compounds that you are looking at.

My suggestion is to use an Atlantis HILIC silica column, equilibrate it in 95% acetonitrile, then inject your analytes with a gradient to at least 50% acetonitrile, followed by an elution of your aminoglycans with formic acid in 50/50 water acetonitrile. You will need to figure out, if 0.1$ formic is enough, or if you need to work at a higher concentration to elute the aminoglycans.

This assumes that your detection tool is MS and that you are interested in the separation of the sulfated species, and not interested in the aminoglycans. If this is wrong, the procedure needs to be modified.

Dear Uwe Neue,

Thank you for your suggestion. However I fear I wasn't clear enough about the nature of my substrates. Glycosaminoglycans, despite their name, do not contain any amino functionality, in fact they are acidic and negatively charged due to the presence of sulfate groups (either on hydroxy or amino groups of the oligosaccharides). I have read that the Atlantis HILIC column is most suitable for polar bases because of the presence of negative silanols. Do you think my oligosaccharides could still be retained on a silica HILIC column ?
Or is it maybe better trying an Atlantis dC18 column?(As far as I know GAG oligosaccharides are not retained on standard C18 unless with ion-pairing)
Many thanks again, I really appreciate your opinion.
Regards,

Emiliano

For HILIC you do not need any amine function. It is partioning into a water layer on the surface of the silica. Any carbohydrate will love to get into this layer, even more so if it has additional ionic functions.

I apparently was not clear: the proposed gradient is exactly what you need to elute neutral and acidic analytes. The second step was a suggestion for cleaning the column from strongly adsorbed basic contaminants, which apparently you do not have.