4.1. Concept and principles.
There are things we don't know we don't know.
D. Rumsfeld
Compared to pFI where the advantages of flow programming are now well documented, this chapter is based on what we know, leading to research about what we don't know, by exploring similarities between the flow-based methods.
Seen from a conventional viewpoint FI and Chromatography are dissimilar because their modus operandi (sample/reagent interaction versus sample/column separation) as well as their instrumentation, are very different. Yet they share a common ground since they use continuous constant forward flow to transport sample from injector to detector where sample components are quantified. Thus, seen from the viewpoint of chemical reactor engineering (Levenspiel 1972, Flow Injection Fluidics) Flow Injection and Chromatography are impulse-response techniques and therefore there are opportunities for flow programming to enhance performance of assay protocols that include column separations.
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cFA is an impulse-response technique based on injection of sample as a rectangular input, that results in a response that yields quantitative information on content of target analytes. There are two types of modulators: reactor, column, and their combinations.
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However, because separation columns are operated at forward flow, they must be included in programmable flow system on opposite site of injection point.
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pFChromatography is an impulse-response technique based on injection of sample as a rectangular input, that travels upstream from injection point into a reactor where target analyte is processed. Following flow reversal moves reaction mixture through a column, where sample components are separated due to difference in migration velocities, resulting in a multicomponent response curve that yields quantitative information on content of target analytes. The flow system can be configured in two ways: as sequential injection chromatography (SIChrom), driven by a single pump, or programmable flow chromatography (pFC), driven by two pumps that move sample through confluence point by flow reversals. Separation of analytes takes place on column placed between confluence point and flow cell.
In analogy with pFI configurations SI manifold can be configured with single pump for Sequential Injection Chromatography (SIChrom) or with two pumps for programmable Flow Chromatography (pFChrom). While single pump construction is simple SI configuration has limitations discussed in Section 3.1.4.
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The key component of two pump system is the confluence point where sample merges with reagents at turbulent flow conditions (Section 3.2.1.). In pFI format this configuration enables techniques which can not be performed in any other way, such as fully automated
While application of flow programming offers opportunities for further improvement of performance of column-based techniques, there are limitations due to properties of stationary phase, column constructions, and auxiliary components.
[1] Šatínský D., Solich P., Chocholouš P., Karlíček R. "Monolithic columns - A new concept of separation in the sequential injection technique" Anal. Chim. Acta, 499 (2003), 205-214.