A Rapid and Precise Method for Nitrate Determination using a Step-Flow Autoanalyzer with Online Stopover Vanadium Reduction

This study presents the development and performance evaluation of a Step-Flow Autoanalyzer that integrates online vanadium-(III) reduction with the Griess assay for the high-precision and efficient determination of dissolved nitrate in aqueous samples. The system manifold comprises seven microperistaltic pumps controlled by six electric relay modules. The nitrate sample is loaded, mixed with reagents, and held in a heating coil at 90 °C for 70 s to facilitate the reduction of nitrate to nitrite. It then passes through a cooling bath, lowering the temperature to below 60 °C to stabilize the pink azo dye color before being delivered to a 1 cm dome-type flow cuvette installed in a spectrophotometer. Absorbance at 543 nm is measured under static conditions. High precision is achieved (<0.5% RSD at the 10 μM level), with a detection limit of 0.1 μM, equivalent to 0.003 absorbance units (AU). The calibration curve remains nearly linear up to 50 μM, covering the concentration range of most environmental samples. The apparent reduction efficiency (E%), estimated by comparing responses from equivalent concentrations of nitrite and nitrate, is approximately 99% in freshwater and 92% in seawater. Each measurement cycle takes 120 s, allowing for a maximum throughput of 25-30 samples per hour. The system's high precision and reliability during extended operation make it well-suited for routine applications involving large numbers of samples.