Sequential Injection (SI), the second generation of FIA techniques, is based on flow programming  (Ruzicka & Marshall 1990). An important milestone in development of SI was its micro miniaturization into Lab-on-valve format, and  its use  as a platform for Bead Injection technique discussed in Chapter 3.

Most recent developments, fast SI and sample split technique, for the first time described in this Chapter, make Sequential Injection the most advanced and efficient flow based analytical method

SI is based on injection of a sequence of sample and regent zones into a stream of a carrier solution.  In the simplest configuration, the single interface method, a sample zone (red) and reagent zone (blue) are injected next to each other into a carrier stream of water (light blue).

During this process, sample and reagent zones disperse within each other, while the reaction product (yellow) is formed on their interface. A flow through detector records changes in a desired physical parameter when the reaction product reaches the flow cell. The instrument operates in stop/flow mode and therefore the system is idle, not using reagents and generating waste, unless a sample is being analyzed.

Recent advances  of SI technique exploit  flow programming to adjust flow rate for optimization of the individual steps of assay protocol. This, along with technological advances in temperature control and pump design, allows, for the first time, to achieve, the same sampling frequency as with FI – over 120s/hr for one reagent  assays (single interface method (2.1.1.A.),  and over 100s/hr for two reagent assays (sandwich method). (2.2.25.)

Sample splitting technique allows exposure of  selected portions of a processed sample, to different treatments, which yields multiple information such as e.g. simultaneous assay of two analytes (nitrate & nitrite) using  a compact apparatus comprising of only one valve and one pump (2.2.22.).

Digital sample processing, low regent consumption and versatility of the method, made  SI a widely used tool  for a research and for continuous monitoring. It will be interesting to see how fast Sequential Injection will replace the Flow Injection as a tool for serial assays, as this will lead to abolishment of continuous flow techniques (and  peristaltic pumping), that still dominate the field of flow analysis.

J. Ruzicka & G.D. Marshall, Anal. Chim. Acta, 237, 329, (1990)