Miniaturized chromate-free chloride assay in fish sauce based on linear height calibration of AgCl precipitate in inverted microtubes

Abstract

• Chromate-free, miniaturized argentometric method for chloride analysis • Direct height-based calibration enabled by geometry-controlled microtubes • Instrument-light assay using ruler readout and low-speed centrifugation • Microliter-scale reagents reduce silver waste and energy consumption • Validated green screening of chloride in high-salt food matrices A miniaturized, chromate-free methodology for chloride determination in high-salt food matrices is presented in accordance with the principles of Green Analytical Chemistry. Classical argentometric precipitation is adapted to a microvolume, instrument-light assay by confining silver chloride formation within inverted microcentrifuge tubes and directly measuring the height of the compact precipitate layer using a simple ruler. Because the cylindrical tube region has a constant cross-sectional area, precipitate height is directly proportional to chloride concentration, enabling straightforward linear calibration without optical detection, signal transduction, or complex data processing. Controlled tube inversion and low-speed centrifugation ensure that precipitation occurs exclusively within this cylindrical zone, producing a sharp, reproducible solid–liquid interface. Under optimized conditions (150 µL sample volume and 180 µL of AgNO₃ in nitric acid; centrifugation at 600 rpm for 3 min followed by 5 min settling), the method provides a linear working range of 50–500 mM chloride (r² = 0.9988), a detection limit of 14 mM, and relative standard deviations of 3–4% at 200–300 mM. Validation of 12 commercial Thai fish sauce samples showed no statistically significant differences compared to AOAC potentiometric titration at the 95% confidence level, with recoveries of 100–103% and %RSD values of 1–4.2%. The primary advantage of this method is its strictly linear height–concentration relationship, enabled by geometry-controlled confinement of the precipitate. Unlike other miniaturized or paper-based assays, it eliminates the need for image analysis or device fabrication. From a green chemistry perspective, the assay eliminates the use of potassium chromate indicators and disposable paper-based devices, operates at the microliter scale using reusable plastic microtubes, and requires only low-speed centrifugation. Analytical Greenness (AGREE), Analytical Eco-scale, Green Analytical Procedure Index (GAPI), and White Analytical Chemistry (WAC) evaluations (AGREE score: 0.76; Eco-scale: 83; WAC score: 88.9) classify the method as an excellent example of green analysis. The proposed approach offers a robust and sustainable screening tool for chloride determination in high-salt foods.