Most studies dealing with fermentation use a Aminex or Rezex column to analyse FVAs, alcohols and some sugars. The method is simple and relatively robust, but in some cases there are peaks that overlap (especially when working with glycerol fermentation).
Some suggest to lower the temperature to better separate glycerol from fumarate, for instance, but will this be enough to separate all the peaks?
Hi Cristiano,
This is a good question! In reverse phase separation, we usually have a number of tools to affect selectivity: gradient changes, buffer selection, column oven, etc.
With the Rezex (as well as chiral stationary phases), we generally have only one mobile phase to work with. In the case of Rezex, the only mobile phase we have to work with is 100% aqueous (water).
In this case (as well as chrial) the only way we can 'tease out' selectivity is with a different stationary phase or ligand. Which Rezex column are you using? Perhaps I can recommend an alternative Rezex, that will give you a different selectivity (and hopefully separate out those coeluting peaks).
Paul
Dear Paul,
thank you.
I have been using the ROA Organic Acid column (300mm), in order to detect the main fermentation metabolites and the substrate consumption (using RI and UV detectors)... it's very helpful when you need to run a lot of samples every day and you want to constantly monitor your process.
So with this column, more than changing the temperature and maybe the concentration of H2SO4 of the mobile phase I guess there is not much else I could do.
Do you agree that lowering the temperature from 65°C to 30°C might already solve some coelution problems?
Dear Christiano,
Do you now the pKa- value of your analytes / impurities? And which pH has your eluent? If the pKa and molecule size (because the separation mechanism is ion size exclusion chromatography) of you analytes are quid similar, then it will be challenging with this type of column
Regards
Matthias
The pH of the eluent should be around 2.5 (the eluent being 5 mM H2SO4).
The pKa of the analytes can be certainly obtained from literature.
In case the pKa difference should be sufficiently large (> 2 ?) what would you suggest to achieve a better resolution of this isocratic method?
Hi Cristiano,
OK, Rezex ROA is the best for the type of work you are doing. Changing the temperature might help a bit. In gerneral, this approach changes the viscocity of the solvent... if the solvent isn't as responsive to changes in temperature, you might not see much of a difference.
One thing you might try: regeneration of the column. Over time, the column may have lost some of it's 'selectivity'. Do you have the regeneration procedure available? If not, please see attached file. This might bring back some retentivity and get you better separation of your peaks.
Paul
Yes, I have done the regeneration several times before (the procedure is in the leaflet provided with each new column..). I might also check if the number of theoretical plates have significantly decreased ...
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