A majority of colorimetric assays uses two or several reagents, which in SI format will have to be assembled in the sandwich sequence, for which the injected volumes of sample and reagents must be properly selected.
The reason is that SI operates as a single stream system, where it is necessary to select the injected sample volume in such a way, that a sufficient concentration of reagent Cr is available to form a reaction product. Therefore the volume of the sample zone (red) should not exceed 3 S1/2 values (A) of the flow manifold. (Chapter 0) since in such case the Ds value will approach 1 and concentration of reagent Cr will approach zero (B). Precise value of S1/2 can be obtained by injecting a dye as a sample zone in the selected sandwich configuration, using such a large sample volume, that a steady state is reached. In the next experiment the injected sample volume is decreased, until a peak is obtained, where at peak maximum Ds value is at least 2. (A,B). Alternatively as a rough approximation, the volume of the channel through which the center of the injected sample will travel to the flow cell can be estimated as one S1/2 value.
Two Reagent Assay
2.2.17.
