Determination of phosphate in freshwaters by flow injection with immobilized enzyme and chemiluminescence detection

Abstract

A flow injection method for the determination of phosphate in freshwaters is described using immobilized pyruvate oxidase based on luminol chemiluminescence (CL) detection. Hydrogen peroxide released by the enzymatic reaction of pyruvate in the presence of phosphate is coupled with luminol in an alkaline medium generated CL response using cobalt(II) as a catalyst. The calibration graph was linear over the range of 2–10 × 10⁻⁶ M phosphate (r ² = 0.9985) with relative standard deviation (n = 5) 1.8–3.8%. The detection limit was 2 × 10⁻⁷ M with a sample throughput of 60 h⁻¹. The common interferences were removed off-line by treating water samples with iminodiacetate chelating resin. The method was applied to freshwater samples and the results obtained [1.05–1.58 × 10⁻⁶ M (0.10 ± 0.02–0.15 ± 0.01 mg 

 L⁻¹)] were in reasonable agreement with the results obtained using the molybdenum blue method with spectrophotometric detection [1.47–1.89 × 10⁻⁶ M (0.14 ± 0.03–0.18 ± 0.02 mg 

 L⁻¹)]. The immobilized pyruvate oxidase exhibited good operational activity at 30°C for a week and storage stability at 4°C over a period of three months.

Keywords:

• Chemiluminescence
• Flow injection analysis
• Phosphate
• Immobilized pyruvate oxidase
• Hydrogen peroxide
• Freshwaters

Acknowledgements

The author would like to acknowledge the financial assistance of the Ministry of Science and Technology, HEC, Pakistan, Prof. Paul. J. Worsfold, School of Earth, Ocean and Environmental Sciences and Plymouth Environmental Research Centre, University of Plymouth, UK for providing research facilities and his valuable suggestions.