Since each step of a flow based assay is best performed at a different flow rate, flow programming is an important component for optimization of reagent based assays. Theá SI protocol for reagent-based assays uses the following combination of flow rates:
WASH fast flow rates for flush the system with carrier solution
INJECT sample and reagent zones at flow rates no higher than the injected volume
REACT for SHC protocol uses stop flow, for SFC protocol uses volume (return volume), which will place the reacting zone within the flow cell at aná intermediate flow rate
MONITOR for SHC protocol uses volume that will push the peak maximum into the flow cell, using a moderate flow rate to collect data. For SFC protocol stop the flow for a desired time interval.
For adsorption of analyte on a microcolumn use very low flow a column (1 to 5 ÁL/sec). For analyte desorption from microcolumná use low flow rates (5ÁL/sec)