Sequential Dilution on-line (Section 2.2.6.G.) provides sample conditioning, that allows the SFC reaction rate enzymatic assay (2.2.11.A.) covering the range of 0.05 to 0.60g/ L glucose to be applied for monitoring in the range 0 to 4 g/L glucose.
For this purpose, the assay was modified by: 1) diluting glucose reagent 1:3 with DI water, 2) sequencing small volume of sample with a large volume of reagent into the holding coil (A) and 3) using assay protocol with 100 sec stop flow period (B red box).
The reaction rate measurement was carried out by arresting the reaction mixture within the flow cell for period of 100 seconds. By setting the baseline readout (SB green box) at 40 seconds and final readout (WIN yellow box) at 120 seconds of elapsed time, the linear portion of reaction rate curve was bracketed (C) and used to construct the calibration plot (D).
While SFC-RR method offers lower sampling frequency (25s/hr) then SHC- end point measurement, it is more reproducible, and also more selective since variations of baseline (red box on recording C) do not influence the calibration plot. This feature is useful when analyzing real life samples, if their matrix /background color interferes with the wavelength at which the target species is being monitored.
Sequential Dilution for Glucose Assay by SFC
Reaction Rate Measurement
2.2.11.D.
