Widely used, constant flowrate based FI, is simple to perform. To optimize sensitivity and sampling frequency of the cFI assay, the length and thus the volume of the conduit between injector and detector have to be adjusted to provide the incubation period required by chemical reactions.
Since peak height, and sensitivity of assay decreases with sample zone dispersion the length of the conduit (A) between injector and detector should be minimized.
Adding reagents at confluence points, shown here at equal flow rates (B), is a key feature of Flow Injection, since it delivers at an even concentration of reagent along the entire length of sample zone.
Increasing the volume of the injected sample (C) increases peak height linearly until the volume reaches one S ½ (equal to D=2), higher S ½ will cause reagent deficiency, unless reagent is added in confluence mode. (B).
Because S ½ value is equal to the volume of channel between injector and detector, it follows that minimizing volume of this channel is a key to miniaturization of Flow Injection manifold. (1.2.24.)
The challenge in designing of cFI system, lies in providing necessary incubationtime, available for chemical reactions to proceed and to produce species to be monitored by a detector. Since for serial assays it is desirable to achieve a sampling rate of at least 1 sample /min, the residence time of a sample in the flow channel is limited to 60 seconds, unless the purpose of an assay is to reach high sensitivity and low detection limit– the usual trade off for reagent based assays.
Surprisingly and fortunately, a vast majority of chemical reactions applied to (bio) and chemical assays, will convert a sufficient amount of target analyte into a measurable species within 30 seconds or less - even at a room temperature.
Fast reaction rate is one of the reasons why the majority of 23.000 entries in Hansen’s database are methods based on continuous flow. The success of cFI made it a workhorse of research and of routine analysis in agriculture, environmental and oceanography, where the most frequently analyzed species are phosphate, nitrate, nitrite and ammonia.