July 07, 2022, 04:04:12 AM
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Topic: Reverse phase HPLC for polar molecules, or the HILIC method ?  (Read 253 times)

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Offline PharmaStud.

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I am working on the determination of 5-fluorouracil and its degradation impurities (Barbituric acid, 5-hydroxyuracil, and Urea). I have to assay these compounds by the same method. The method that I found in the Pharmacopoeia for the determination of impurities (apart from urea) is reverse phase liquid chromatography. However, if I have understood correctly, reverse-phase HPLC is for non-polar molecules, and for polar molecules the normal-phase HPLC method or the HILIC method should be preferred.

I have several questions on this topic:

1) Why is the reversed phase used in most scientific articles and in the pharmacopoeia but not the HILIC method or normal phase HPLC. I have searched in vain but I can't find anything as an explanation. I even read in an article that 5-fluorouracil cannot be assayed by reverse phase HPLC because it is an extremely polar molecule. So i'm a bit lost.

2) Why is urea not measured by this method? And is this molecule measurable by the reverse phase HPLC with a UV detector? Because I see in certain articles written "Urea does not absorb in the UV" and then in the pharmacopoeia I see that it can be assayed by HPLC at a wavelength of 190 nm with a UV detector.

3) What method would you recommend for measuring all these molecules? I was thinking of using the HILIC method, but I'm a bit lost, I'm still a student and it's the first time I'm developing a method so I'm afraid of doing it wrong.

It would help me a lot if someone could answer my questions,
Thanks in advance.

Have a nice day.


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Re: Reverse phase HPLC for polar molecules, or the HILIC method ?
« Reply #1 on: April 17, 2022, 10:34:10 PM »
I am not a chromatographer per se, but I know that UV at 190 nm is so-called "end absorption", pretty much every compound will absorb at 190.

The trick then is to use a mobile phase that absorbs minimally at 190 nm.

Others are more qualified to answer your other questions.


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