09 September 2015 6 8K Report

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

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