Analysis of Urea
Fast, Reproducible HPLC Method
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Figure above shows 5 consecutive runs using 100% water mobile phase without any loss of
||Cogent Bidentate C18, 4µm, 100A
||4.6 x 150 mm
||A: DI water (ISOCRATIC Run: 100% A)
||Urea (1mg/mL in DI water)
||UV 210 nm
A Cogent Bidentate C18 column was successfully used to retain Urea when a 100% DI water
mobile phase was used. Since this hydrophobic column was made on TYPE-C Silica DI water
can be used as the complete mobile phase without fear of phase “dewetting” and subsequent loss
of retention time. The presented method has the advantage of being very simple, without time
consuming derivatization or reaction steps when compared to other methods using ordinary C18
columns. Urea can be determined in complex mixtures using this very fast, reproducible (see
insert on the chromatogram) method which could also be useful in the analysis of biological
samples for diagnostic purposes.
Note: There is growing demand for a sensitive and reliable procedure for the
determination of urea in many matrices such as milk, soil extracts, seawater and wine.
In addition there are several clinical applications for the analysis of this compound.
The most common approach for measurement of urea involves detection of ammonia
(after hydrolysis) by color forming reactions – enzymatic, colorimetric methods. The
other techniques require noxious reagents and produce an unpleasant odor .
Newer methods involve high-performance thin layer chromatography-densitometry,
alkalimetric titration. HPLC is the most specific method but either organic normal
phase chromatography or ion pairing chromatography have to be used to retain this
very polar compound until this method.
 “Determination of urea using HPLC with fluorescence detection after automated
derivatization with xanthydrol”, S. Clark, P.S. Francis, X.A. Conlan, N.W. Barnett, J.
Chromatography A, 1161 (2007) 207-213.