A practical method for sensitive determination of the fluorescent water-tracer uranine by reversed phase HPLC under alkaline conditions

A stable and highly sensitive HPLC method for uranine has been developed. Because of unstableness of silica-based octadecyl-C18 columns at high pH condition, a reversed phase HPLC analysis under alkaline conditions has not necessarily taken as a usual method. However, the application for uranine seems to be advantageous, since the fluorescence yield of uranine is markedly enhanced at high pH condition. The detection limit of the HPLC system was 0.9 pg. The analytical consideration was also paid for the solid phase extraction (SPE) prior to the HPLC analysis with careful consideration of the recently revised pKa values of uranine. The recovery rate of uranine by SPE was found to depend on the sample volume and a few ml of seawater was applied to SPE in order to maintain the recovery rate during SPE. A combination of HPLC and SPE methods achieved detection of uranine at concentrations as low as 0.2 ng l−1 (0.5 pM), which was comparable to the background concentration of uranine in coastal water off Japan. For the practical use of the detected tracer-uranine concentration values after substantial duration after release, the photodegradation of uranine in surface water was also evaluated in terms of incident solar radiation dose as an exponential rate constant of −0.135 mol photon−1 m2.