SPECTROMETRIC DETERMINATION OF PHOSPHATES IN SOIL

Background

Most phosphorus in the soil is found in the form of inorganic salts, however it is also present in organic compounds.

The limited value of annual entry is 120 kg P₂O₅ per hectare.

The content of P₂O₅ in soil according to the AL method and range limits:

level	mg P₂O₅/100 g soil	condition of soil
A	<6	poorly
B	6–12	middle provided
C	13–25	well (goal reached)
D	26–40	excess
E	>40	extremely

Principle of experiment

All the phosphates present in the soil sample are transformed either by a wet or dry decomposition into orthophosphates, which yield a yellow colour upon addition of molybdene salts. After reduction with hydroquinone and sodium sulphite the product becomes blue. The intensity of the blue colour is measured by a spectrometer with a red LED.

Labware/Equipment

Laboratory spoon
Laboratory bottle 500 mL
Volumetric flasks 50, 100 and 1000 mL
Bulb pipettes 5 mL
Funel
Blue filter paper

Automatic pipettes 20–200 µL

Blisters
Magnetic stirrer
Magnet
Analytical balance
Spectrometer Spektra^TM

Reagents

Ammonium molybdate solution: 5 g of anhydrous ammonium molybdate or ammonium molybdate tetrahydrate is dissolved in 60 ml of deionized water. In parallel 15 ml of concentrated sulphuric acid is diluted with 40 ml of deionized water and this solution is added to the former solution. The final solution is then kept in a dark reagent bottle in a dark place.
0.5 % solution of 1,4 dihydroxybenzene (hydroquinone): 0.5 g of hydroquinone is weighed into a 100 mL measuring flask and filled to the mark with deionized water. One drop of concentrated sulphuric acid is added to prevent oxidation. The solution is kept in a dark reagent bottle in a dark place.
20% solution of Na₂SO₃: 20 g of Na₂SO₃ is weighed into a 100 mL measuring flask, dissolved with deionized water and filled to the mark with deionized water. The solution must be freshly prepared.
A standard solution of K₂HPO₄: 0.2210 g of K₂HPO₄ is weighed and quantitatively transferred into a 1000 mL measuring flask, 1 mL of 0.1 M H₂SO₄ is added and the solution is filled to the mark with deionized water. 5 mL of the solution is pipetted into a 50 mL volumetric flask and filled with deionized water to the mark.
0.1 M ammonium lactate: 5.17 mL of 20% solution ammonium lactate (Sigma-Aldrich, 377228) is pipetted into a 100 mL volumetric flask and filles with deionized water to the mark.
0.4 M acetic acid: some deionized water is filled into a 100 mL volumetric flask, then added 2.29 mL of 100% acetic acid and filled with deionized water to the mark.

Hazards

	Sulphuric acid is corrosive, irritating to eyes, respiratory system and skin. It causes severe burns. Protective clothing, eye/face protection and gloves should be used. In case of contact with the eyes, immediate rinsing with water and a visit to the doctor are necessary. R: 35 S: 26-30-36/37/39-45
	Hydroquinone is a hazard for health and the environment. It should be kept in a closed container. Suitable protective clothing, gloves and glasses should be used. R: 22-40-41-43-50 S: 26-36/37/39-61

	Ammonium molybdate is a hazard for health. It irritates the eyes, respiratory system and skin. In case of contact with the eyes, immediate rinsing with water and a visit to the doctor are necessary. R:36/37/38-52/53 S:26-36-61
	Sodium sulphite is a hazard for health. It irritates the eyes and skin. In case of contact with the eyes, immediate rinsing with water and a visit to the doctor are necessary. R:22-31-36/38 S:26-36/37
	Ammonuim lactate: no hazard. If inhaled, remove to fresh air. In case of skin contact immediately wash with copious amounts of water. In case of contact with eyes, flush with copious amounts of water. If swallowed, wash out mouth with water.
	Acetic acid is corrosive, flammable and causes severe burns. If inhaled, remove to fresh air. In case of skin contact, wash with plenty of water. Immediately call in opthalmologist. After swallowing drink water, call in physician. Do not attempt to neutralize. R:10-35 S:23-24-45

Procedure

Sample preparation
The sample is prepared using the AL method with extraction. A 5 g representative sample is sifted through a sieve (hole sizes 2mm) and weighed into a laboratory bottle. 100 mL of ammonium lactate and acetic acid (1:1) and a magnet is added to the sample bottle. The bottle is put on a magnetic stirrer and stirred for 2 hours at 200 RPM at 20 °C . After stirring the sample is immediately filtrated through the filter paper into a 100 mL volumetric flask. The flask is filled with deionized water to the mark and well stirred.

Determination of the phosphates
The procedure, shown in the table, both for the blank probe and for the sample is followed.
An adequate volume of the sample is pipetted into the blister. The transmittance is measured after 30 minutes relative to the blank probe with a red LED.

	sample (µL)	V_P₂O₅ (µL)	V_H₂O (µL)	V_Hydroq. (µL)	V_Molybd. (µL)	V_Na-sulph. (µL)	T (%)	A	c (mmol/L)	γ_P₂O₅ (g/L)	m_P₂O₅ (µg)
Blank probe	-	-	160	80	80	80			-	-	-
Calibr.sol.1	-	20	140	80	80	80	98,8	98,8	0.00403	0.00057	0.2288
Calibr.sol.2	-	40	120	80	80	80			0.00806	0.00114	0.4576
Calibr.sol.3	-	60	100	80	80	80			0.01209	0.00172	0.6864
Calibr.sol.4	-	80	80	80	80	80			0.01612	0.00229	0.9152
Calibr.sol.5	-	100	60	80	80	80			0.02015	0.00286	1.144
Calibr.sol.6	-	120	40	80	80	80			0.02418	0.00343	1.3728
Calibr.sol.7	-	140	20	80	80	80			0.02821	0.00400	1.6016
Calibr.sol.8	-	160	-	80	80	80			0.03224	0.00458	1.8304
Sample	160	-	-	80	80	80

Calculation

The calibration line is a function of absorbance versus mass of P₂O₅. The mass of P₂O₅ in the sample is deduced and the mass fraction of P₂O₅ in the original sample is calculated by the following equation:

m_P₂O₅ = m_{P₂O₅ (cal.line)} ×	100
	0.16

m(P₂O₅/100g) =	m(P₂O₅)×100g
	m(sample)

Developed and prepared by: Janja Dvoršek, Tadeja Polajnar, Kristina Frlic, Biotechnical centre Naklo