1. Monitoring of phosphate in natural waters by using an automated multi-pumping pulsed flow system with spectrophotometric detection Marta F. T. Ribeiro a,b, Cristina M. C. M. Couto a,b, Pedro M. M. Conceição a, João L. M. Santos b a CICS, CENTRO DE INVESTIGAÇÃO EM CIÊNCIAS DA SAÚDE, INSTITUTO SUPERIOR CIÊNCIAS DA SAÚDE- NORTE, GANDRA, PAREDES, PORTUGAL b REQUIMTE, DEPARTAMENTO DE QUÍMICA, FACULDADE DE FARMÁCIA, UNIVERSIDADE DO PORTO, PORTUGAL Introduction In this work, an automated multipumping pulsed flow system [1] was implemented for the determination of phosphate in natural waters. The developed flow approach was based on the spectrophotometric determination of phosphate by using the vanadomolybdate reaction [2]. The exploitation of a very simple manifold configuration relying on the utilization of just two active components, in this case two solenoid actuated micropumps that were accountable for sample and reagent insertion and commutation, reaction zone formation, and solutions propelling, provided a great operational and optimization simplicity, low reagent consumption, and waste minimization. The system was applied to the monitoring of phosphate in local streams at specific sampling stations. Figure 1 – Location of the sampling stations within the main streams of the Porto city, Portugal. S1 and S2 - Abade and Gramido streams (crossing rural areas), S3 and S4 - Tinto river 1 and 2, and S5 and S6 - Granja stream 1 and 2 (crossing more industrial and urban areas). Flow System Figure 2 – (1) Flow manifold of the proposed MPFS system: S, sample; R, vanadomolybdate reagent; P1 and P2: solenoid micro-pumps; X, confluence point; L, reaction coil (50-cm); D, detector (420 nm); W, waste (2) and (3) Picture of a solenoid micro-pump (10 µL per stroke) and the developed flow system, respectively. (3)(2) P2P2 P1P1 X D L W (1) S R FLOW MANIFOLD OPTIMIZATION Sampling Strategy (X, Figure 2) Merging zones Reagent Volume (R, Figure 2) 50 μL 100 μL 150 μL 200 μL 250 μL 300 μL 50 cm Reactor Length (L, Figure 2) Ammonium Vanadate Concentration (R, Figure 2) Ammonium Molybdate Concentration (R, Figure 2) Hydrochloric Acid Concentration (R, Figure 2) 0,010 M 0,015 M 0,5 M FIGURES OF MERIT Table 2 – Figures of merit for the determination of phosphate in the developed flow system and in the reference procedure (APHA) [2] ParameterFlow System APHA Procedure Linear regression a A = 0.023(±0.001) [P]+0.001(±0.004)- Correlation coefficient (r 2 )0.999- Linear range a 0.5-201-18 Detection limit a 0.2 Assay time1 min10 min Reagents consumption per assay: Ammonium molybdate4.6 mg250 mg Ammonium vanadate2.9 µg12.5 mg Concentrated hydrochloric acid0.01 mL3.3 mL Estimated amount of waste produced per assay 1.5 mL50 mL a mg L -1 PROCEDURE APPLICATION TO FIELD SAMPLES Table 1 – Analytical cycle StageProcedure Solenoid micro-pump Pulse number Pulse time (s) 1 Baseline establishment P1950.6 2 Reagent introduction P1+P2250.6 References: [1] João L.M. Santos, Marta F.T. Ribeiro, Ana C.B. Dias, José L.F.C. Lima, Elias A.G. Zagatto, Anal. Chim. Acta 600 (2007) 21. [2] American Public Health Association (APHA), Standard Methods for the Examination of water and wastewater, 20th ed., APHA Press, Washington, DC, 1998. Acknowledgments: The authors thank CESPU the financial support through the Project Ref. 03-GCQF-CICS-10. Conclusions Site Proposed Method Reference Method Relative error (%) S13.50±0.213.42±0.02 2.34 S26.03±0.016.39±0.02 -5.63 S37.59±0.367.58±0.15 0.13 S47.70±0.437.58±0.14 1.58 S54.38±0.114.27±0.20 2.58 S62.51±0.152.33±0.05 7.72 Table 3 – Comparative results. Phosphate concentrations (mgL -1 ) as determined by the developed flow system and the APHA reference procedure The developed multipumping pulsed flow system could be a valuable strategy for the determination of phosphate in superficial and wastewater samples. It can be an advantageous alternative to other available procedures, because it exhibits a high degree of automation, it is simple, fast, precise, accurate, requires low reagent consumption and minor operator intervention.