Maximizing the slope of calibration line and minimizing the blank value optimizes sensitivity of an assay and improves limit of detection. The blank value of PMoB method is composed of reagent blank (MoB) and analyte blank of phosphate, present as impurity in the reagents used. Ideally, spectrum of PMoB will exhibit a well defined peak at 880nm while spectrum of BLANK will be almost flat above 550nm (A).
It is the combination of acidity with molybdate concentration [H+]&[Mo] in presence of antimony, ascorbic acid and surfactant which determines sensitivity as well as reagent blank. The assay protocol (B) and corresponding script (C) is designed to mix ascorbic acid with molybdate 1+1 (step 2) and sample with mixed reagents (3+1). The molybdate and ascorbic acid reagents must be prepared in such concentrations that will yield in the flow cell an optimized reactant mixture: [Mo]=1.0mM, [H+]=0.3N HCl, 0.03mM KSb tartarate as well as 43mM AA and 0.75% SDS) (1.5.8.)
It follows from [H+] diagrams (1.5.10.) that various [H+]&[Mo] combinations will yield optimized conditions and that in addition to hydrochloric acid sulfuric and nitric acids are suitable.
Hydrochloric acid is used here in stead of commonly used sulfuric acid, because it can be efficiently purified by isothermal distillation that will eliminate analyte blank.
Calibration is here performed in SFC mode using incubation time of 30 seconds and absorbance collection within 59-64 second time frame (WIN).
The calibration line, obtained with 20cm long light path flow cell yields sensitivity and LOD suable for trace determination of phosphate in sea water (LOD=30nM P). Although there is more work to be done, our previous work based on the same SFC methodology and instrument (Hatta et, al, 2019) indicates that this method has potential to become useful for determination of phosphate in sea water. Further increase of sensitivity is achieved by preconcentration of PMoB by sorbent extraction (3.2.5.)
M. Hatta, Ch. I. Measures, J. Ruzicka, Talanta 191, (2019) 333.