Photometric Determination of Ammonium and Phosphate in Seawater Medium Using a Microplate Reader

Abstract

To more efficiently process the large sample numbers for quantitative determination of ammonium (NH₄⁺) and phosphate (orthophosphate, PO₄^3-) generated during comprehensive growth experiments with the marine <i>Roseobacter</i> group member<i> Phaeobacter inhibens</i> DSM 17395, specific colorimetric assays employing a microplate reader (MPR) were established. The NH₄⁺ assay is based on the reaction of NH₄⁺ with hypochlorite and salicylate, yielding a limit of detection of 14 µ<smlcap>M</smlcap>, a limit of quantitation of 36 µ<smlcap>M,</smlcap> and a linear range for quantitative determination up to 200 µ<smlcap>M</smlcap>. The PO₄^3-assay is based on the complex formation of PO₄^3- with ammonium molybdate in the presence of ascorbate and zinc acetate, yielding a limit of detection of 13 µ<smlcap>M</smlcap>, a limit of quantitation of 50 µ<smlcap>M,</smlcap> and a linear range for quantitative determination up to 1 m<smlcap>M</smlcap>. Both MPR-based assays allowed for fast (significantly lower than 1 h) analysis of 21 samples plus standards for calibration (all measured in triplicates) and showed only low variation across a large collection of biological samples.