Flow Injection Tutorial

In order to optimize conditions for automation of this assay, the rate of formation of PMO was investigated first, using assay protocol and reagent composition described in the previous page, by changing WAIT time to adjust the incubation period of the reaction mixture held in HC2, thermostated at 25 C. (A). Surprisingly, maximum absorbance was reached at 0 sec stop time, while longer incubation periods yielded decreasing absorbance values. This decrease in absorbance is probably due to formation of colloidal phosphomolybdenum blue, which is sparingly soluble in water.

Sensitivity of all FI assays increases with injected sample volume and therefore, as expected, by increasing volumes of 400ppB P standard, an increase of peak height was experienced (B). Note, however, that the peak height is limited at Sv of 100mcrL by selection of the transfer volume from HC1 to HC2 in the first step of the assay protocol.

Calibration by means of serially diluted standard solutions yielded responses in the range of 0 to 800ppB P, shown superimposed (C). The calibration graph (D) was obtained by collecting peak height measurement in a window (WIN) and by using data from BS interval for baseline subtraction.

Comparison of reproducibility between miniSI (2.2.25.) with with pFI with average rsd 4.2% for phosphate assay (D), at similar calibration ranges confirms feasibility and reliability of pFI when applied to two reagent SHC protocol.