Method 59a Determination of extractable phosphorus manual method

The following manual procedure is based on MAFF/ADAS, 1986, pp. 183-185 (with Crown Copyright Permission).

Phosphorus is extracted from soil at 20 ±1°C with a solution of sodium bicarbonate at pH 8.5. The absorbance of the molybdenum blue complex produced by the reduction with ascorbic acid of the phosphomolybdate formed when acid ammonium molybdate reacts with phosphate is measured using a spectrophotometer at 880 nm.

Reagents (extraction).

• Polyacrylamide solution, 0.05% m/v - dissolve 0.5 g of polyacrylamide in approximately 600 ml of water by stirring for several hours. When dissolved, dilute to 1 l.

• Sodium bicarbonate reagent - dissolve 420 g sodium bicarbonate (sodium hydrogen carbonate) in water, add 50 ml of the polyacrylamide solution and dilute to 10 l. Add approximately 50% m/m sodium hydroxide solution, stirring with a glass rod, until the pH meter reading is steady at 8.50 at 20°C (a plastic Pasteur pipette is useful for dropwise addition approaching the required pH).

Procedure (extraction). Transfer 5 ml (scoop filled and struck off level without tapping) of air-dry soil, sieved to ^2 mm into a bottle (e.g. wide-mouth, square HDPE). Add 100 ml of sodium bicarbonate reagent, pH 8.50, cap the bottle and shake on a reciprocating shaker, at approximately 275 strokes of 25 mm length per minute, for 30 min at 20°C. Filter a portion immediately through a Whatman No. 2 filter paper, rejecting the first few millilitres of filtrate. Carry out a blank determination.

Reagents (determination).

• Ammonium molybdate reagent, 1.2% m/v - dissolve 24 g powdered ammonium molybdate (ammonium paramolybdate, (NH4)6Mo7O24.4H2O), and 0.6 g antimony potassium tartrate (Note, cumulative poison) in 1200 ml water. Slowly add 296 ml sulphuric acid (approximately 98% m/m H2SO4), stir slowly with a glass rod, and dilute to 2 l. (Note: sulphuric acid is highly corrosive and generates heat when diluted; standing the beaker in a sink with a few centimetres depth of cold water before adding the acid will reduce any likelihood of localized boiling. Wear PPE for this step.) Store in a dark glass bottle in a refrigerator.

• Ammonium molybdate reagent, 0.15% m/v - dilute 1 vol. of 1.2% m/v ammonium molybdate reagent to 8 vol. with water. Store in a dark glass bottle in a refrigerator.

• Ascorbic acid solution, 1.5% m/v - prepare immediately before use, allowing 5 ml per standard, blank and sample, with some spare for any repeats.

• Phosphorus stock standard solution, 1 mg P ml-1 - dry potassium dihy-drogen orthophosphate at 102°C for 1 h and cool in a desiccator. Dissolve 0.879 g of the dried salt in water and add 1 ml of hydrochloric acid, approximately 36% m/m HCl. Dilute to 200 ml and add 1 drop of toluene to the solution.

• Phosphorus intermediate standard solution, 20 pg ml-1 - pipette 10 ml of the phosphorus stock standard solution, 1 mg ml-1, into a 500-ml volumetric flask, make up to the mark and mix. Add 1 drop of toluene to the solution.

• Phosphorus working standard solutions, 0-7 pg P ml-1 - prepare fresh daily solutions by pipetting 0, 5, 10, 15, 20, 25, 30 and 35 ml of the phosphorus intermediate standard solution, 20 pg ml-1, into 100-ml volumetric flasks, make up to the mark with sodium bicarbonate reagent, and mix. These will contain 0, 1, 2, 3, 4, 5, 6 and 7 pg P ml-1 respectively.

• Sulphuric acid, approximately 1.5 M - slowly with stirring, add 80 ml sulphuric acid, approximately 98% m/m H2SO4, to about 800 ml water in a 2-l beaker (Note: sulphuric acid is highly corrosive and generates heat when diluted; standing the beaker in a sink with a few centimetres of cold water before adding the acid will reduce any likelihood of localized boiling. Wear PPE for this step.) Cool and dilute to 1 l.

Procedure. Pipette 5 ml of each phosphorus working standard solution (i.e. 0, 5, 10, 15, 20, 25, 30 and 35 pg P) into a plastic vending cup (or 100-ml conical flask). Add 1 ml of approximately 1.5 M sulphuric acid and swirl the solution to assist the release of CO2. Add 20 ml of 0.15% m/v ammonium molybdate reagent, 5 ml of ascorbic acid solution, 1.5% m/v, swirl to mix and allow to stand for 30 min for colour development. Measure the absorbance in a 10 mm optical cell at 880 nm. The colour is stable for several hours. Construct a graph relating absorbance to pg P. The absorbance values should be approximately 0 to 0.8 for the 0 and 35 pg P standards respectively.

Similarly pipette 5 ml of the soil extract into a plastic vending cup, followed by 5 ml sulphuric acid, 1.5 M. If the soil extract solution is highly coloured after addition of the acid, pipette a duplicate sample and add the 5 ml of sulphuric acid, 1.5 M. Add the other reagents, as detailed above, to the first sample, but only add the ammonium molybdate reagent to the duplicate. Measure the absorbance at 880 nm. For coloured extracts, subtract the absorbance of the duplicate without ascorbic acid, which will not develop the blue colour, from the absorbance of the sample extract with ascorbic acid.

Calculation. Read from the standard graph the number of pg of P equivalent to the absorbances of the sample and blank determinations. Subtract the blank from the sample value, and multiply the difference by 4. The result gives the mg l-1 extractable phosphorus in the air-dry soil. This can be expressed for oven-dry soil as in Method 5.2, Calculation (2).


1. If one of the standards produces an absorbance that lies significantly away from the standard graph produced by the other standards, or if the whole graph is erratic, repeat as necessary. Detergents containing phosphates should be avoided, but ones such as Decon 90 are phosphate-free.

2. The use of phosphate-free carbon to decolorize soil extracts has been found to give erratic results.

3. A slightly less accurate determination is possible using a colorimeter with a wide bandpass filter, e.g. a simple (non-interference type) purple-red filter but an interference filter in a quality instrument gives results comparable to a spectrophotometer.

0 0

Post a comment