Soluble Reactive Phosphorus
Soluble Reactive Phosphorus
Use filtered water samples (Whatman GF/F glass-fibre filters preferred). Method also suitable for TP samples following digestion.
- Sulphuric acid: add 70ml conc H2SO4 to 450 ml DDW
- Ammonium molybdate: dissolve 1.5 g of (NH4)6Mo7O24.4H2O in 50ml DDW (do not use if more than 6 weeks old)
- Ascorbic acid: dissolve 2.7 g of ascorbic acid in 50 ml DDW [make up fresh (very gentle warming may be required)]
- Potassium antimonyl tartrate: dissolve 0.34 g in 250ml DDW [make up fresh (gentle heating may be required)]
- Standard phosphate solution (50 mg/l): dissolve 0.2195 g of oven dried KH2PO4 in DDW and make up to 1000 ml (in the Lab fridge)
Prepare the mixed reagent in a fixed ratio as follows - reagent (a) 5 : (b) 2 : (c) 2 : (d) 1, mixed in that order - Make up fresh. 1 ml required per sample
i.e. For 100 ml mixed reagent: 50 ml: 20 ml: 20 ml: 10 ml
For 50 ml mixed reagent: 25 ml: 10 ml: 10 ml: 5 ml
For 20 ml mixed reagent: 10 ml: 4 ml: 4 ml: 2 ml
Where the concentrations of PO4-P are greater than 10 µg/l, samples and standards can be prepared using lower volumes. Prepare 10 ml volumes of the samples and the standards in disposable centrifuge tubes and add 1 ml of the composite solution; cap and invert the tube several times to mix. This method is particularly suitable when larger numbers of samples are being analysed. The laboratory centrifuge tubes are sterile and P free.
In practice this method has been found to provide accurate and repeatable results on samples with PO4-P concentration in the range 5.0-1000 µg/l. It is however recommended that samples with very high PO4-P concentrations are diluted with DDW to below 200 µg/l PO4-P.
Take 1.00 ml of standard phosphate solution (50 mg/l) and make up to 100 ml to produce a working P stock solution of 500 µg/1 as P.
A set of standards must be treated simultaneously to the water samples. Set up the standards using the following dilutions of the 500 µg/1P stock solution.
- 10.00 ml DDW (0 µg/L PO4-P). This is the reaction blank
- 9.50 ml DDW: add 0.50 ml P stock (25 µg/l PO4-P)
- 9.00 ml DDW: add 1.00 ml P stock (50 µg/l PO4-P)
- 8.00 ml DDW: add 2.00 ml P stock (100 µg/l PO4-P)
- 7.00 ml DDW: add 3.00 ml P stock (150 µg/l PO4-P)
- 6.00 ml DDW: add 4.00 ml P stock (200 µg/l PO4-P)
1. Place 10.0 ml of the filtered water samples into 15 ml centrifuge tubes
2. Add 1.00 ml of the mixed reagent to each tube (including the standards), cap tightly and invert to mix.
3. After 0.5-12 hours measure the absorbance in a quartz cuvette at 885 nm against the reagent blank (0 µg/L PO4-P).
Where the concentrations of PO4-P are very low (e.g. <5.0 µg/l), larger sample volumes should be used (200 ml) and an additional step is required whereby after the composite solution is added the blue complex is extracted into an organic solvent (e.g. isobutanol) in a separating funnel.