The colorimetric assay of phosphate developed in the previous section is based on a two step reaction. First o-phosphate reacts rapidly with ammonium molybdate (MO) forming yellow phosphomolybdate which, by a catalyzed reduction with ascorbic acid (AA), forms molybdenum blue colloid, monitored at 880nm. In this experiment aimed at high sensitivity, spectrophotometry is carried out using 10 cm light path flow cell (A, B). Therefore the assay protocol is redesigned to assemble the sandwich using relatively large sample and reagent volumes, in order to assure that the flow cell (volume 50 μL) is fully charged with the reacting mixture for the reaction rate measurement. Selection of the reversal volume (400μL) (2.2.11.) and stop flow periods (25 sec and 120 sec) was done by methods described previously (2.2.18.).
By using stop flow period of 25 seconds (C) the linear calibration covered range 5 to 100 ppB P. Using 120 second stop flow (D) the sensitivity and LOD improved 10 times, the detection limited being due to reagent blank (15 nM P). The sample throughput was 45 s/hr, and 22s/hr.
High Sensitivity P Assay
2.2.19.
