Dear Esther Dijkstra

Try to change the pH of the elution buffer

Dear Esther Dijkstra try to find the solution via the followed files

Article Microwave-assisted Dilute Acid Pretreatment and Enzymatic Hy...

Dear Esther Dijkstra

Using an Amino column under the conditions, you described means that you are running HILIC conditions. I am a big fan of HILIC, but sometimes it is a bit tricky. First: In HILIC acetonitrile (ACN) is the weak eluent, while water is a strong eluent. Hence, the samples must not be present in a pure aqueous solution. Why? Because if they are, you will co-inject the strong eluent, which in other words can and will cause the separation to "collapse." Hence my first suggestion is to prepare the standards with a high concentration of organic solvent (ACN).

Your move towards higher ACN concentrations is a good one, as this should improve the separation - remember ACN is the "weaker eluent."

Then: You might not like the following: If your samples contain reducing sugars, the amino columns tend to age, due to the formation of what is known "Schiff-base." Hence, how old is the column, and was it in use before for the analysis of sugars?

Consider the column temperature as well. Usually, we chromatographers tend to think in "the higher, the better" when it comes to temperature. Which in most of the cases is true, for all the benefits of reduced backpressures, higher efficiencies due to increasing diffusion processes... There is however a BUT...

But there are many examples out there where cooling the column (or generally speaking using the column at lower temperatures) had significant benefits. Keep an eye on the column backpressure as viscosity will increase with decreasing temp, but you should be okay with the high ACN concentrations.

Finally, HILIC is one way of separating sugars. And w/o knowing better, I'd assume that you are after higher concentrations (RI-detector!). You can use this detector also in conjunction with other separator columns like ligand exchange columns. Those are cation exchangers loaded with different cations (H, Ca, Ba, Ag). You can have such columns from different vendors, but maybe you find such a column in your institute.

A good starting point for method searches is this free database:

https://appslab.thermofisher.com/

I did a quick search for Carbohydrates and HPLC, and there are several interesting HILIC entries.

I hope this is of some help.

Good luck with your research

Detlef.

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