Flow Injection Tutorial

There are not many options left for further increase in sensitivity of this assay, since elevating of temperature, prolongation of incubation time, and of the length of the

light path, have reached practical limits.

Yet, since the temperature of the flow cell (A) is so well stabilized, and assay protocol (B) is so well controlled by software, it turned to be feasible to expand the calibration range down to low ppB level (C) by monitoring reliably the change of absorbance within the range of 0.1 A unit (D), and thus obtain a calibration graph (E) with LOD of 3 ppB N. The reagent background signal although high, (0.3 A) turned to be remarkably reproducible, provided that the reagents were prepared fresh daily.

Note that the response curves are result of two processes; heating of the reaction mixture from

21 C to 45 C during the initial lag phase, followed by the reaction rate curve, when indophenol blue is being formed. This approach, of keeping HC at room temperature, was chosen with aim to maximize response, since, if the reaction was initiated already in a heated holding coil, the amount of product formed within the flow cell would have been smaller, and the sensitivity of the assay lower.