Hi everyone! I am currently working on an HPLC method to try to get clean peaks for 8 different volatile fatty acids. Almost all of them are clearly identified in my results, but there are two isomers (isovaleric acid and 2-methylbutyric acid) with peaks that almost completely overlap with the method I'm using.
Column: Hi-Plex H, 4.6 x 250 mm made with sulfonated styrene/divinylbenzene matrix in hydrogen ionic form, 8% crosslink, 8 μm particle size, designed for acid conditions at 40-60°C.
Column: 35C
Refractive index: 35C (not using UV, just RID)
Injection volume: 5uL,
Flow rate: 0.2mL/min
Mobile phase: 5mM sulfuric acid isocratic
*I have also tried runs where I've increased the concentration of H2SO4 mobile phase to 10mM and another where I've tried to increase column temp (55C) and RID temp (50C) and this did not appear to help the separation*
Based on the literature, it seems pretty tough to separate isovaleric and 2-methybutyric acid without the use of MS or another technology (which we do not have access to). Can someone tell me if this is likely futile tweaking my current method with this column and set up? Do I need other equipment instead?
Ion exchange HPLC with your current setup is unlikely to separate isovaleric acid and 2-methylbutyric acid isomers effectively. Achieving clean separation typically requires alternative columns, chiral selectors, or advanced detection like mass spectrometry.
Abdelhak Maghchiche I figured that might be the answer. Thanks for the response! Is there an alternative column that comes to your mind that might be a better fit for this particular situation?
Your current ion-exchange HPLC setup is unlikely to separate isovaleric acid and 2-methylbutyric acid isomers effectively. For clean separation, derivatization combined with GC-MS or LC-MS/MS using reversed-phase or specialty columns is recommended.
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