Composition of reagents used here, is based on the work of Hatta (2018), except that molybdate was combined with ascorbic to form a single reagent (A). This change simplified the assay protocol and allowed to use lab-on-valve manifold based on six position valve (B). The microcolumn (10mm long, 1.6mm I.D.) sandwiched between LOV module and 10 cm long flow cell was packed with 200 microliters of Stata XL sorbent. Suspension of sorbent beads was stored in a syringe attached to port #6. Microcolumn lasted for a large number of measurements and was replaced about once a week.
The influence of temperature on formation of PMoB was investigated by assay protocol set to one loading, stop time 40 seconds and by using 5 ppB P standard solution (C). Based on this observation all further experiments were performed at 50 C.
The influence ofincubation time was investigated by changing stop time (WAIT period in step #3 of the assay protocol), while using 5 ppB P standard solution (D). Note however, that actual incubation time, in addition to stop flow period, was longer, because 1000mcrL volume of reaction mixture was delivered from the holding coil at a rate of 25mcrL/sec. Stop time of 40 sec was selected for all following experiments.