Chromatography Forum

Instrument: Agilent 1100 series with UV/RID detectors

Column: BioRad Aminex 300mm

Mobile phase: H2SO4 in H20 @ 0.6 mL/min


I'm running simultaneous sugar/organic acid (as well as a particular diol) analysis on fermentation broths and the current run time is too long (25 min). I was just wondering what other methods (columns ect.) you guys were using for this type of analysis?

thanks!

I don't have an easy answer. I don't think there is one. Options 2 and 3 are fastest but option 1 has the lowest activation energy.

One option which would work for you would be to derivatize then use reversed phase and your UV. Many schemes are available. $

Another option is to use the same material PS-DVB in a 50 mm long format (available from Phenomenex as a guard) and use MS in negative mode detection. $$$

A third option is to go with an ELSD and run them by HILIC mode. There are a number of vendors for columns but I prefer Tosoh amide and the new Waters 1.7 um amide phase. $$

I agree with mardexis. I would start with HILIC first. Please check out the application data bases of different manufacturers which are available online, and ask for a free test column. HILIC columns are available also in smaller ID and shorter column length, but first try the standard dimension to see if you get the resolution you need.

The problem with HILIC is you can't do both organic acids and sugars via this methodology. You could use the fermentation Bio-rad column it is shorter uses the same mobile phase but has a shorter run time. Also, your current separation is not as sensitive to dissolved solids or pH changes and HILIC might be.

You should give more information about what organic acids and sugars you are looking for - as well as moble phase separation.

You might want to consider something like this:
http://www.sielc.com/application_183.html

I have added a reference, which will show that HILIC is indeed a tool for both sugars and organic acids.

Metabolic profiling of intracellular metabolites in fermentation broths from β-lactam antibiotics production by liquid chromatography-tandem mass spectrometry methods
B. Preinerstorfer, S. Schiesel, M. Lämmerhofer, W. Lindner
J. Chromatogr. A, 1217 (2010) 312-328

"Employing a silica based zwitterionic stationary phase in the HILIC mode, in total 223 hydrophilic metabolites can be determined. In particular,
amino acids, organic acids as well as nucleotide sugars were found to be well separable and detectable under acidic mobile phase conditions, while in comparison especially phosphates such as nucleotides,
coenzymes or sugar phosphates as well as sugars and sugar acids performed better at higher pH."

Yes like Phycal mentioned I am limited to UV and RI detectors. Using an isocratic mobile phase of water and H2SO4 (5mM) I am able to observe sucrose, glucose, fructose ethanol and butanediol on the RI detector while simultaneously detecting organic acids (pyruvic, succinate, formate, lactate, Acetic) on the UV 210nm.

As I mentioned before the method is robust (basically no sample prep) but analysis time is 25 min, which makes my analysis time in days...

Perhaps I will try this short Biorad Aminex column...