Hello everyone,
I've been using an HPLC for VFA detection but I have little experience. I'm specifically looking for acetate, propionate, butyrrate, valerate, etc. . I'm working with a c18 column and gradient eluition (water/methanol), DAD set at 210 nm.
One of my main concern is that I got different results in chromatograms even in "simple" standard tests: for example, I did several runs of pure acetic acid, to get a "standard" peak, but I got slightly different times at the end of the runs(I have to say that this acetate is not HPLC quality).
How can I make more consistent my analyses? My main goal now is to make a library of VFA standards, and later analyse mix of VFAs.
Thanks
Hi.
On a first glance, I would say you need an aqueous eluent with a pH of about 2 - 3, not deinonised water. This will keep you acids protonated thus allowing you to maximize the efficiency and reproductability of the chromatographic separation on your RP column. It the pH of your mobile phase is close to the pKa of the analytes, then they will be partially deprotonated and small variations in the conditions will cause significant variations as the ones you observe. If the pH of your mobile phase is higher than the pKas of your analytes, then they will just come out with the injection front because they will be fully ionised, thus not retained by the column.
Cheers!
Hi, Mário Silva ,
thanks for your answer! I have to say that my aqueous phase was added of sulfuric acid, but in a very small concentration(0.005 mol/L), so I don't think this had a big impact on the overall pH.
But you think adding the correct amount of sulfuric acid until ph 2~3 to HPLC water is ok to get my correct aqueous phase?
Thanks,
Maurizio
My advice is for you to check the pKa of your analytes and keep the pH of the mobile phase bellow the lowest one. Also, you could try isocratic (with low fraction of methanol) instead of gradient elution. A strong retention of your analytes on RP C18 is not likely.
Cheers!
Mário Silva ,
thanks again for your answer, I will try as soon as possible with this indications.
Best regards
Dear Maurizio Cuomo
Many thanks for your question. There are much better columns available for the analysis of volatile organic acids than a C18 column. Due to their high polarity and low hydrophobicity, low molecular weight organic acids are no good target group for C18.
One could try HILIC or mixed mode (anion exchange + RP) for the separation. The latter has the advantage that the HILIC mode can be switched on/off as well.
However, the real deal for organic low molecular weight acids, w/o touching the great alternative of modern anion exchange chromatography, is the use of Ion Exclusion Chromatography. Use an aqueous, acidic eluent and then use UV (@ 200 - 210 nm). Alternatively, RI-detection is applicable as well. Both UV and RI, have one thing in common: They are not the most sensitive detection schemes for aliphatic organic acid. As long as you are after higher concentrations, you are fine. Lower concentrations (single/double digit mg/L-range) are accessible with conductivity detection, usually applied as what is known as "suppressed conductivity". Yes, in conjunction with Ion Chromatography Exclusion. Lower levels are accessible with ion exchange chromatography, and in many cases, gradient elution is applied.
A great source of information and a starting point for further evaluations might be this free of charge applications database called AppsLab:
https://appslab.thermofisher.com/
You will find mixed-mode columns from different vendors, as will you find Ion Exclusion columns as well. I think, if you are continuing to use a conventional HPLC instrument (stainless steel), then have a look at ion exclusion chromatography. Easy to apply and very robust. I would start there.
I hope this helps.
Good luck with your research.
Best regards,
Detlef.
Both (maybe more) Waters, Agilent and Phenomenex have methods for VFAs or organic acids. Check their websites!
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