Chromatography Forum

Hello
I separate several fermentation products with an aminex H+ (HPX 87H, BioRad) column. Under standard conditions, 5mM sulfuric acid, 0.5 ml/min and 50oC, the separation is very satisfactory.
My problem is now with acetoin.
The acetoin standard runs at 20.45 min, and its spectrum on DAD shows two maxima at 200.7 and a smaller one at 275 nm.
The fermentation samples show two peaks, the first is at 19.2 and shows the same spectrum as compared to the standard. The second is at 20.33 min and its spectrum is very different to the standard as well as the first peak.
Both, standard and sample are prepared not in mobile phase, but in water or culture media respectively, then filtered and injected.

My question is : Is there a possibility that the acetoin in my sample (and not in the standard) suffers some transformation due to the mobile phase?

Thanks

It is far more likely that you are seeing another compound in addition to acetoin.

Yes, Tom... I agree with you.
What has intrigued me is that the spectrum shape of the first peak is, overlapped with the standard spectrum, equal. I understand that this might happen, as DAD is not so specific. But here the only thing I can be certain of is that I can't report any of these peaks as acetoin.
Thank you

My question is : Is there a possibility that the acetoin in my sample (and not in the standard) suffers some transformation due to the mobile phase?

I'm fairly new to this area, but I wanted to share my fermentation chem experience. My old lab tested fuel ethanol fermentation samples looking for acids that may indicate a bacterial infection at different locations in the plant. We were also using 0.5 mM H2SO4.

I had some chomatograms that had terrible resolution in the Sugars area, but all my system parameters were good, and my check standards ran fine. To make a long story short: samples that were badly infected with Lactic Acid bacteria could contain up to 3% lactic acid. This was enough to overwhelm the acid in the mobile phase. I was able to determine the lactic acid was able to distort the glucose peak so badly that it lost it's identity.

If you have any suspicions about sample contaminents like bacteria waste products or cleaning residues etc. Try getting some pure chemical to spike your standard with and run it as a sample. If there is some interaction going on you may be able to identify what it is, and determine if it is a problem worth trying to fix, or not. Also, do you purchase the mobile phase premixed? Sadly, There can be a lot of variation between technicians. It might be worth buying it pre-mixed just to reduce the potential variation.

Good Luck!

Thank you Jade.
Good idea, I will spike my samples.
Let me tell you that your comment on your experience surprises me a little. We have a project which involves lactic acid overproducing bacteria, fed with glucose and/or xilose as carbon source. Our lactic (as the predominantly main product) production levels are in the same order of magnitude, around 50 g/l, and without dilution, we were able to measure sugar consumption withou problem. I believe that the used medium (minimum), almost as working with standards, could have been the difference.

Can acetoin exist as 2 tautomers?

Likely, but they would interchange so fast that one would not separate them in LC.
The original problem could be due to a different interference with the chromatography. If I understood the question correctly then hdz.geo injects the standard in H2O, but the unknown in culture media.
How does this work? The acetoin is (partially) protonated? The culture media is stuffed full with ions which displace the acetoin?