I use HPLC to measure FAD to FMN conversion by my enzyme. The conditions are as follows:
A: 15 mM formic acid, pH 4.5 by NH3
B: MeOH
column ZORBAX Eclipse Plus C18 RRHD 2.1x50mm 1.8 micron
in the beginning the pH of MF A was 4.0 as we used that for other compounds and it worked for FAD and FMN as well. However, later it was shifted to 4.5 due to some ghost peaks in other analyses.
After some previous problems
https://www.researchgate.net/post/A_new_peak_appeared_in_HPLC_that_should_not_be_there_What_could_be_the_reason I wanted to try, whether pH lower than 4.5 would improve peak shape/resolution. Thus I tried pH 3.5, but the FAD peak disappeared completely. , so I tried pH 4.0 and the peak was there, but very broad and mis-shaped.
After I switched back to pH 4.5, the peak was OK, but in comparison to previous analyses it is slightly shifted later (2.1 vs. 2.4 min), but mainly it's very broad (width at 50% is more than twice it used to be), the tailing factor is about the same.
So I have two questions
1) why the pH 4.0 doesn't work when I used it in the past routinely?
2) why is the peak shifted and so broad?
Thanks in advance
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention.
There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
The problem relates to the method itself. Formic Acid's pKa is 3.75. Your mobile phase pH is near the pKa of the acid so small changes in pH will result in large changes to peak shape and retention.
There are many excellent books and papers on HPLC method development, mobile phase choices and buffer selection. If you perform a keyword search, you will turn up plenty! However, one guide that I can recommend which will explain the importance of buffer choice, acids and pKa values plus their effects on retention times can be found at this link [ http://www.hplc.eu/Downloads/ACE_Guide_BufferSelection.pdf]. *Ignore the promotional adverts and read the sections on HPLC buffer choice. I think they provide a simple explanation. Hope this helps!
It looks like you're just pH adjusting and not buffering. You might be better served having 15 mM formic acid + 15 mM ammonium formate and adjusting to your pH's to ensure your pH stays constant.
I find that my shifts are due primarily to insufficient equilibrium time after my gradient returns to starting composition. If you're just looking for 1 peak, find an isocratic method that gives you good S/N, and then really push the peak through with as high of flow as your LC will tolerate to minimize broadening. If your injections are dirty enough that you want to start with high water for rinsing the column and junk away first, then eluting with high organic to clean the column of organics, you'll need a long equilibration for good T(r) precision. I'll usually return back to initial composition as quickly as I can after my peak of interest comes of to minimize time, but it does depend on the concoction you're injecting. The greater the gradient difference, the longer the equilibration time. Choose as short, narrow-bore, and small particle size of a column as you can to minimize this exchange volume.
Monitor your LC pressure profile. Make sure the pressure curve is smooth and returns to the starting pressure during your re-equilibration.
Thanks William, that seems interesting.
@Stephen with all respect, it really doesn't matter, whether you mix acid and base or acid and salt if you're in the right pH range. The equilibrium will be always the same (except I have 15 mM formate anion, while you'd have 30 mM formate).
I'll check the equilibration, but the method is still the same, so it shouldn't change.However, shouldn't it cause frontal tailing?
Careful pH adjustment, stability of pH and sufficient equilibration may be crucial in certain methods as William and Stephen already pointed out. Other than that, column aging can cause peak broadening worsening by time up to peak splitting with a method that has previously worked. You can try a fresh column of the same kind or run the column backwards to rule it out.
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