HPLC Method for Quantification of Urea

[fargherm]

Hi all,

I wanted to know if any has used a specific method for quantifying urea.

Currently I am doing:

Dissolving pure urea in 90:10 Acetonitrile:Buffer (ammonium acetate, acetic acid, pH 5)

Method:

Mobile phase- 93% acetonitrile 7% buffer
Column- NH2
UV- 200, 210, 230
RI Detector
Inj Volume- 5 uL
Flow- 1 mL/min
Column Temp- 40

So far I have not even been seeing the urea. I have made up many concentrations and nothing seems to have an effect. There is a peak near 5 on the RID and DAD, but it never changes with different concentrations. The detector is not saturated and neither is the solution.

Any ideas or experience would be greatly appreciated.

Thanks!

[Andy Alpert]

"Determination of urea, allantoin and lysine pyroglutamate in cosmetic samples by hydrophilic interaction chromatography" - Ph. Dallet et al., J. Chromatogr. B, 742 (2000) 447.
The authors used a PolyHYDROXYETHYL A column (200x4.6-mm; 5-µm, 60-Å) with 6 mM triethylammonium phosphate (TEAP), pH 2.8, and varying amounts of ACN. It looks like they settled on 88% ACN. Detection was via absorbance at 200 nm.

Commenting on your method: Acetate ion has a much higher molar extinction coefficient than does urea at all of the wavelengths that you are monitoring. TEAP was used in the above paper because it's transparent at 200 nm. You can have a mobile phase that's transparent at low wavelengths or one that's volatile, but not both. An exception is one with TFA, but that antagonizes retention in the HILIC mode. When you're performing HILIC of urea, you need all the retention that you can get.

[fargherm]

Thank you! I will look into that. I have seen that the NH2 column could cause quite a bit of baseline noise (which is present).

I may try to use a phosphate buffer instead of the ammonium acetate if I can't seem to pinpoint any other problems.

[Karen01]

An Aminex HPX-87C column with RI will work for urea . Mobile phase : Water @80C 0.6 ml/min.

- Karen

[juddc]

I'm taking this from memory, so it may be less than perfect, but I've assayed for urea without much difficulty in a relatively simple matrix using an amino column (Shodex NH2P50, I think). I believe my MP was 80/20 ACN/0.1% H3PO4 with detection in the low UV.

Alternately, I would also predict that it would be assayable via reversed phase with ELSD detection if using a perfluorinated ion pairing agent (TFA, HFBA). I've assayed for highly polar urea derivatives that way pretty easily.

CJ