pFI: Single Reagent Assay by SHC; Nitrite  
One Transfer
The key feature of this protocol is the stop flow period in its second step (A) at which reaction mixture is arrested to provide incubation time, defined by the WAIT period of the software script (B). The Gries reaction, which is the basis of nitrite assay ( see 1.2.32.),  was used to test feasibility and precision of this, single transfer, single reagent protocol. By changing duration of incubation time (C) it was established that WAIT of 5 seconds is sufficient to generate adequate yield of the monitored dye. The influence of the injected sample volume (D) confirms, similarly to cFI technique, that the peak height increases with increasing Sv until reaches limiting value (at 100mcrL in this case). Calibration run (E) shows six superimposed response curves, similar to the conventional cFI peaks. The calibration graph (F) was obtained by  automated data collection,  and by plotting difference of A values between baseline (BS) and peak maximum  located in the window (WIN). It follows that the single transfer SHC method has and average rsd of 1.1% a reproducibility better than experienced by majority of cFI or miniSI assays.