Hello! I‘m a student and the topic of my research is developing a method for quantitive analysis of glucosamine and chondroitin (in tablets after dissolution) via HPLC-DAD.
We‘ve been using tosyl chloride for derivatisation and NaOH for hydrolysis of chondroitine. After analyzing glucosamine and chondroitin (after hydrolysis and derivatisation) separately and together, we got just one peak. It seems that those are derivatized glucosamine and galactosamine together.
We tried using gradient mode, changing pH and mobile phases, but it didn’t make things better at all. I also tried ion-paired additives (octanesulfonate), but I was told that it can destroy the column so I stopped. Is it even possible to separate these two compounds with a C18 column?
I would be very grateful for your advice!
Thank you in advance!
Equibilirate the ODS column using cationic ion pairing reagents (such as alkylated quaternary amine salts) and convert all the aminosugars into their anionic form by increasing pH. In slightly alkaline conditions (be careful not to exceed the column pH resistance), this methodology presents IEX interaction (AEX). Isomeric sugars may be separated using an ODS column employing an IEX bind and elution approach. The reverse may also be beneficial, with anionic ion pairing and an acidic environment (protonated amine) version, but I predict AEX would be more promising. Look to HPAE-PAD applications for easy method transfer...
Dear Alexandra Tretyakova
I am not sure that RP chromatography is the way to go. HILIC might be something to try - or a mixed mode WAX/RP or WAX/HILIC. Imho, though, I would suggest HPAE-PAD - like Ismail already did. To boost this statement, please find a paper published by my colleagues linked below.
You find all the experimental data, the does and don'ts, described in great detail.
All the very best for your research
Detlef
https://www.thermoscientific.com/content/dam/tfs/ATG/CMD/CMD%20Documents/Application%20&%20Technical%20Notes/Chromatography/Ion%20Chromatography/IC%20and%20RFIC%20Columns/77324-AN233-HPAE-IC-Heparin-29June09-LPN2286.pdf
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