Flow Injection Tutorial

Since all reagent-based assays were originally designed for batch type format in which all reactions must attain chemical equilibrium, descriptions of assay procedures offer no information on reaction rates (Marczenko 1986). Therefore, with the exception of enzymatic assays, one is usually left in the dark about the minimum time needed to obtain a reliable readout. Colorimetric assay of phosphate by molybdenum blue method is an example. The classic batch procedure prescribes 10 min reaction time at almost 100 C (Marczenko 1986). The AutoAnalyzer method is based on 3 min reaction time. In cFI format a reliable readout is obtained within 20 sec (Ruzicka 1975), and the recently developed accelerated protocol for FI assay of requires less than 10 sec to obtain a reproducible readout.

Considering that it took more than 30 years to optimize automation of one of the most frequently performed assays it is not surprizing that “translation ” a many classical reagent-based assay into FI mode, is far from being optimized , and that many existing FI (or SI) protocols could be further improved through investigation of reaction kinetics.

Therefore, when we evaluate a classical reagent-based assay as a candidate for automation by FI or SI, we should not be discouraged by incomplete or misleading information on the time frame needed for its completion. Indeed, the focus of future research and its novelty lies in a thorough exploration of the complex kinetics of the reactions, which are the basis of reagent based assays, because such work will discover surprising findings about their speed , and about reaction rate based discrimination which will reveal conditions for improved selectivity.