Sequential Injection technique, is based in the same principles as FI: sample injection, controlled dispersion andreproducible timing. The difference is that SI uses flow reversals in a programed sequence to control these parameters.
Controlled dispersion takes place as stacked zones move upstream into the holding coil and then move back through the valve into a detector, during the flow reversal. This process forms a well-defined concentration gradient that is seen as a continuum of elements with varying concentrations of analyte, product and reagent. To produce a readout that is proportional to the initial concentration of the analyte, it is essential to achieve complete overlap of sample by reagent zones. The overlap is evaluated by measuring the dispersion coefficient of the sample (D = C° / Cmax) as it yields a degree of sample dilution. Since the concentration of reagents is always higher than that of the analyte at D>2, there will be sufficient reagent excess in the reacting mixture. mp must be added between the valve and the holding coil to provide a reagent confluence point. Zone sequencing is discussed in the following sections.
Reproducible timing in an SI system is achieved through repeatability of all events. This includes sequencing of sample and reagent into the holding coil, transport of stacked zones to the detector and length of the stop flow period. Therefore, T is the time elapsed from the moment of injection (T°) to the moment of peak maximum readout (Tmax) at the end of the stop flow period.
Sample and reagent injection provides the initial input, serving as a starting point for the initial concentration (C°) of analyte (red) and reagent (blue). The volume of injected sample must not exceed one S1/2 value of the programming sequence, corresponding to a D value more than 2.