Non-specific binding could be responsible either between the polysaccharide and the adsorbent or between polysaccharide molecules. Does the polysaccharide have a hydrophobic moiety? Wash the system with an acid solution and see if you desorb the remaining polysaccharide. Wash with a surfactant solution and see if it desorbs.

Dear Marcelina,

as Omar already pointed out, the strong unspecific interactions of the carbohydrates in your sample with the sephadex resin may be caused by hydrogen bonds and weak hydrophobic interactions in addition to the anion exchange effect. I observed this sometimes with proteins or polymers in various chromatography steps leading to delayed or incomplete elution in size-exclusion chromatography and to sample losses in ion exchange or affinity chromatography. The origin of this effect lies most probably in the fact that common chromatography resins are based on derivatized carbohydrates (e.g. sepharose, sephadex...), and often do not behave as inert towards the sample as expected. The ionic strength in the running buffer significantly influences the sample interactions with the carbohydrate part of the resins, and usually high ionic strength reduces unwanted unspecific interactions.

Especially free carbohydrates should show pronounced interactions with a carbohydrate-based resin. Obviously, you cannot add salt in you AEX running buffer which would not only prevent the unwanted interactions, but also binding of your anionic target molecules. What you could try to recover the strongly bound carbohydrate fraction is weakening the presumable hydrogen bonds and hydrophobic effects. High-salt solutions do not help here as they force the carbohydrates out of solution and even strengthen the interactions with the carbohydrate-part of the resin, but chaotropic agents (such as guanidinium chloride, urea,...) or amphiphilic solvents like ethanol (e.g. 20 %) should help in recovering the strongly bound carbohydrates from the resin.

Hope some of this works!

Best,

Joscha

Dear Marcelina,

There is a column chemistry available, called Dionex Cryptand. It has the interesting capability of an adjustable ion exchange capacity. My team successfully used this approach to separate and elute polysulfonated polysaccharides. The chemistry is amazing, and the results are convincing. We used different detection schemes including UV, IPAD, and suppressed conductivity. Please check the web for information. I thought to help you a bit with it and downloaded and attached the column manual to this message.

I hope this helps.

Good luck with your research

Detlef.