OPTIMIZING FI BASED ASSAY
Nothing is more mundane than the FI-based spectrophotometric assay of phosphate, nitrate or ammonia. While vast numbers of these assays are performed in countless laboratories worldwide, no one in Academia, nor any funding agency would give it a slightest consideration as a research topic.
Yet the phospho molybdenum blue method for o-phosphate, the indophenol blue method for ammonia and Gries reaction for converted nitrate are ideally suited to demonstrate the use of flow programming and of other tools, for optimizing the flow based assays. Also there is a documented need for improvement of the throughput and detection limits of these very important assays, since they have many real life applications. And, finally, these methods share features with many other reagent assays, and therefore the following examples will serve as a template for optimization of other flow based methods.
The similarities are:
- use of two reagents in sequence to promote a two step chemical reaction
- reaction kinetics, the second, slower reaction being the rate determining step
- spectrophotometric detection
- identical flow schemes
To begin, we will decrease the volume of the flow path, trough which sample travels on the way to detector by premixing Reagent 1 and 2 outside the sample path:
1.2.28.
Although reagents for phosphate and reagents for ammonia assay must be prepared separately, since their mixture would decompose upon storage, their premixing in this way is feasible, due to the shortness of the residence time on the way to sample stream.
