I have been working on a method for determination of fluorouracil (5FU) (with uracil impurity). I was running on a Phenomenex Prodigy C18 column with water as the mobile phase (USP conditions) and with buffer (low pH phosphate). With the Prodigy column, I obtained equivalent peak shape and retention with either mobile phase for both uracil and 5FU. As discussed in some other current threads, I had problems with variable and decreasing retention times (phase collapse perhaps?).

I moved to a Phenomenex AQUA C18 column with polar endcapping. I ran a mixture of 5FU and uracil with water as the mobile phase. I obtained very nice peak shape and consistent retention times for Uracil, but the 5FU peaks were horribly ill-shapen. I changed to buffer (pH3-7, phosphate, or acetate) and the peak shape of both the 5FU and Uracil were quite nice.

My question is why does the 5FU require buffer (it appears that any buffer - salt or pH - will do) whereas the Uracil does not. With varying pH, the retention times do not change too much which suggests to me that there is not much in the way of ionization happening here. Plus, this would seem to affect both Uracil and 5FU similarly.

Thanks for any ideas.

Kevin

It must be a matter of polarity of the uracils and thus ionic strength of the mobile phase. The columns you use have a considerably different behavior toward polar substances (own experience).
Apparently, uracil exists in the diketo form at pH below 8.5, in the keto-enol form at pH 13, and as the enol at pH above 13. These tautomers have different polarities. The tautomerization should be shifted considerably by a fluorine substituent, which itself can introduce stark differences in polarity.

Maybe we can figure this out if you tell us whatt the polar endcapping of your column is?

Unfortunately, the polar endcapping reagent is not specified in the catalog, I believe it is proprietary.

As far as the polarities go, if I were observing the different tautomers, wouldn't I expect them to have significantly different retention times based on their differing polarities?

Thanks, for your contributions to the discussion.

Normally, tautomers equilibrate very fast, besides, as mentioned before, there appears to be an overwhelming predominance of one isomer at a given pH. The result is that you should see only one peak. In the case of 5FU it may be that your column/mobile phase interferes with an equilibrium and starts to differentiate tautomers at low ionic strengh.
Whatever happens, itīs a very interesting example.