Decreasing glucose and xylose peak area - cause unknown

[magi]

Greetings,

We recently started having issues with our Aminex HPX-87H column chromatography (5mM H2SO4 in water mobile phase). We began seeing a consistent decrease in peak area and peak height over time for the sugars we were analyzing (glucose, xylose and arabinose). We first saw this decrease in one of our HPLCs, but have since started having the problem in all three of our instruments. These decreases occur in conjunction with increased peak tailing and changes in the baseline chromatography. We have tried changing the solvent, changing the column and guard column (the decrease occurs even with brand new columns) and flushing the system to remove microbial contamination. Regenerating the columns helps for a short while, but soon the sugars begin decreasing in concentration again.

Any suggestions as to what may be causing these issues?

[tom jupille]

If your samples contain cations (even something like sodium), those can gradually displace the H+ from the column and give rise to shape problems. Check with BioRan for regeneration procedures (flushing with acid).

[magi]

The samples that we run on this column include acid hydrolyzed biomass water extract, enzymatic hydrolysis of solid biomass, some water extract of biomass, and some fermentation broth. All are filtered at 0.2um before analysis using Watman mini-Uniprep syringeless filters. We have been running these kinds of samples for years without any problems, although we recently switched from syringe filters to the Watman syringless filters. I have tried regenerating the column, after which the chromatography is normal for about 100 runs, then begins to degrade again.

[tom jupille]

If regeneration fixes the problem, which the reappears over 100 injections, *something* in your samples is contaminating the column. Try going back to the old filters temporarily???