Determination of phosphate by the molybdenum blue method is one of the most frequently performed reagent based assays. The PMoB is produced in two steps; the first one results in formation of a polymolybdate structure around the phosphate anion, while during the second step, the yellow phosphomolybdate is reduced to phosphomolybdenum blue:
PMoB based assay is performed in strongly acid solution (pH 0-1) in order to prevent interference from molybdenum blue (MoB). Reduction by ascorbic acid in the presence of Sb (Murphy, Riley 1962), is most frequently used, because it enhances the rate of formation of PMoB, thus making the method suitable for automation by Flow Injection. Spectrum of thus formed PMoB has two maxima at 710 and 880nM, yielding detection limit at micro molar phosphate concentrations. In order to increase sensitivity of PMoB assay, determination of sub micro molar levels of phosphate in open ocean waters is performed either by extending the light path of the flow cell, (Gimbert 2007), or by pre concentrating the PMoB from a large volume of sample by Sorbent Extraction. The latter approach was developed by Heckermann (2000), who in his pioneering work described a Flow Injection method, based on formation of PMoB, its capturing on a polymeric sorbent and elution with sodium hydroxide.
This method is automated here by programmable Flow injection, using miniSIA-2 instrument, configured in the way depicted on pages 3.1.6. and 3.1.7. Protocol for SE-pFI assay, shown in diagram (A) and detailed in software script (B) comprises five steps.
1) reagent is aspirated into holding coil (HC1)
2) valve is turned to sample port and 1000mcrL is aspirated into the second holding coil (HC2), while 200mcrL are delivered from HC1 trough the confluence point resulting in 4+1 sample reagent mixture in HC2. The mixture is incubated for 40 seconds.
3) Valve is turned towards flow cell and reaction mixture is loaded on the microcolumn.
4) eluant is aspirated into HC 2,
5) PMoB is eluted into the flow cell. Finally all channels are flushed with carrier solution. Software protocol shown here is set for two loadings resulting in two accumulation cycles each injecting 800mcrL of sample solution.