Improving the stability of the malachite green method for the determination of phosphate using Pluronic F68

The analytical determination of phosphate constitutes a fundamental step for the evaluation of catalytic activities of many enzymes involved in the hydrolysis of phosphate-containing biomolecules. The most sensitive colorimetric methods to quantify phosphate are based on measuring the spectral changes produced by the adsorption of malachite green to 12-molybdophosphoric acid. Malachite green methods are time-consuming because they require the preparation of color reagent on the day of use, due to its low stability. In this work we propose a modification of the malachite green method that overcomes these problems and only requires a one-step, ready-to-use stock solution including perchloric acid and Pluronic F68. The improved reaction mixture allowed the quantification of phosphate with a limit of detection of 0.22 μM, a dynamical range up to 80 μM depending on the optical path-length, and a molar absorption coefficient at 640 nm of 94000 M^-1cm^-1. Color development reaches a steady level within 30 min and remains constant for at least 2 h. The high stability of the color reagent allows long-term storage for at least 1 year. The optimized procedure is especially useful to measure phosphate-containing biomolecule levels and enzyme activities when low values are critical.