Specific H⁺ level is crucial for accurate phosphate quantification using ascorbate as a reductant

Owing to its essentiality for cellular metabolism, phosphate (PO₄^3-) plays a pivotal role in ecosystem dynamics. Frequent testing of phosphate levels is necessary to monitor ecosystem health. Present investigations were aimed to identify the key factors that are essential for proper quantification of PO₄^3-. Primarily, H⁺ levels played a critical role in the development of molybdenum blue complex by ammonium molybdate and PO₄^3- with ascorbic acid as a reductant. Molybdenum blue complex formed in the presence of 8 to 12 mmol of H⁺ in 3 ml reaction mixture remained stable even after 72 h. Of different concentrations of ammonium molybdate and ascorbic acid tested, best molybdenum blue complex was formed when their concentrations were 24.3 and 5.68 μmol, respectively. More or less similar intensity of molybdenum blue complex (due to reduction of phosphomolybdic acid and not molybdic acid) was formed in the presence of H⁺ at levels ranging from 8 to 10 mmol in 3 ml reaction mixture. Our findings unequivocally demonstrated that (i) the reaction mixture containing 3% ammonium molybdate, 0.1% ascorbic acid and 5 M H₂SO₄ in the ratio of 1:1:1 is ideal for PO₄^3- quantification; (ii) antimony (Sb) significantly curbs the formation of molybdenum blue under these ideal conditions; (iii) this fine-tuned protocol for PO₄^3- quantification could be extended without any problem for determining the level of PO₄^3- both in plant as well as soil samples; and (iv) Azotobacter possesses potential to enhance levels of total PO₄^3- in leaves and grains and soluble/active PO₄^3- in rhizosphere soils of wheat.