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

Abstract

Owing to its essentiality for cellular metabolism, phosphate (PO43-) 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 PO43-. Primarily, H+ levels played a critical role in the development of molybdenum blue complex by ammonium molybdate and PO43- with ascorbic acid as a reductant. Molybdenum blue complex formed in the presence of 8 to 12mmol of H+ in 3ml reaction mixture remained stable even after 72h. Of different concentrations of ammonium molybdate and ascorbic acid tested, best molybdenum blue complex was formed when their concentrations were 24.3 and 5.68μmol, 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 10mmol in 3ml reaction mixture. Our findings unequivocally demonstrated that (i) the reaction mixture containing 3% ammonium molybdate, 0.1% ascorbic acid and 5M H2SO4 in the ratio of 1:1:1 is ideal for PO43- quantification; (ii) antimony (Sb) significantly curbs the formation of molybdenum blue under these ideal conditions; (iii) this fine-tuned protocol for PO43- quantification could be extended without any problem for determining the level of PO43- both in plant as well as soil samples; and (iv) Azotobacter possesses potential to enhance levels of total PO43- in leaves and grains and soluble/active PO43- in rhizosphere soils of wheat.