Anomers being detected? Fructose by CAD detection

[MestizoJoe]

Hi all,

I injected Fructose USP reference standard and I see three peaks. The void, and two peaks, at least one of them must be Fructose.

I developed the method by starting with strong elution conditions (to elute everything) and moved to weaker elution conditions to see peaks retain longer and longer. Starting at 50% buffer and ending up at 7.5% buffer.

Method:

amide column, acetate buffer (7.5%)/acetonitrile (92.5) isocratic; 60C
cad detector
diluent: methanol

Am i seeing two anomers? Is it a closed ring fructose and a open/linear chain fructose?

I'm very confused and the internet searches are not helping.

Thanks!

[HPLC chemist]

It’s unlikely to be an anomer (it may be the solvent peak of Methanol). Try injecting increasingly larger concentrations of fructose. The peak that is proportionately increasing in area is the fructose peak.

[MestizoJoe]

Does it make sense that methanol would retain so long? Void time is one minute, peak 1 is at 6 min, peak 2 at 18 min. If I use either peak alone to calibrate, my check standard recovers within 90% to 110%. Might be a coincidence that both peaks work for standard check, but I took it as evidence of two fructose peaks.

Also when I injected only methanol I don't see the same peaks.

[MestizoJoe]

It’s unlikely to be an anomer (it may be the solvent peak of Methanol). Try injecting increasingly larger concentrations of fructose. The peak that is proportionately increasing in area is the fructose peak.

I injected linearity standards (essentially what you suggested) and saw only the earlier peak behaved like linearity experiment. Thanks!

[Vlad Orlovsky]

from my experience, if you are using wrong pH, peak shape for fructose will show sign of separation of two compounds. You can have a single peak if you go to a lower pH (2.5). used 95% ACN with 0.5% formic acid to generate single peak for fructose (HILIC separation with ELS detection)