Development of an assay should always begin with study of reaction kinetics by using SHC protocol. By increasing temperature of the holding coil, while using fixed stop flow period, influence of temperature is investigated first (A left). Since in this case (ammonia assay) little was gained at higher temperatures, 55 C was chosen to investigate the influence of time by stopping the reaction mixture in the holding coil (A right).
Selection of sample volume for two reagents, sandwich sequence (B), influences sensitivity of the assay, since peak height initially increases, with increasing sample volume, but after reaching a maximum, declines due to lack of reagent within the center of the sample zone.
Sampling frequency. While during method development, it is practical to transport sample trough the flow cell at a slow flow rate (20 mcrL/sec), until all reactants have been washed out (C blue line) the sampling frequency can be accelerated by increasing the flow rate right after peak maximum was recorded. The volume, allowed to pass the flow cell at a low flow rate, is identified by cutting of the trailing edge of the peak. This is achieved by accelerating the flow (to 250mcrL/sec). Using cutout volume of 300 mcrL (D), reduced the sampling cycle from 42 to 33 seconds.
Baseline variations. While the interface between carrier (water) and reagent passes trough the flow cell, baseline changes are recorded due to changes of refractive index (D left) or reagent color (D right). These changes of absorbance, are well time resolved from following peak, and therefore a time window can be defined, within which the software will identify and capture peak height data corresponding to sample composition.