5. Spectrometric determination of phosphates
in meat products

BACKGROUND

Polyphosphates are added to meat cans and salads, to dishes in a jelly , to all meat products that are heat- treated and to all sorts of sausages, except dry and semi-dry sausages. The polyphosphates are added together with nitrite salts.

The amount of the phosphates in the product should not exceed 0.5 % expressed as phosphorus pentoxide.

Upon addition of phosphates the meat proteins can again bind water, and the fats are again capable of emulgation.

PRINCIPLE OF EXPERIMENT

All the phosphates present in the sample are transformed either by a wet or dry decomposition into orthophosphates, which yield a yellow colour upon addition of molybdene salts. After the 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
Crucible
Volumetric flasks (100 ml)
Bulbs (5ml)
Funnel
Blue filter paper
Automatic pipettes

Blisters
Homogenizer
Dryer
Analytical balance
Furnace
Desiccator
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.
HCl in a ratio 1:2 : 25 ml of concentrated HCl is diluted with 50 ml of deionized water.

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
	Hydrochloric acid is corrosive, irritating to the 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: 34-37 S: 26-45

PROCEDURE

Sample preparation

Approximately 100 g of a representative sample of a meat product is weighed and homogenized. 4 to 5 g (±0.0001 g) of the homogenized sample is weighed into a preannealed and weighed crucible. First it is dried in a dryer at 105°C for 2 to 3 hours so that a major part of the water is removed. Then it is heated above the flame of the burner in the fume hood. The sample should not start burning. When the crucible content is charred, the crucible is transferred into a preheated furnace and burned for 4 hours at 525°C, max. 550°C. The ashes should be white to lightly grey- coloured. After the burning the crucible is cooled in the desiccator and weighed.

The total amount of ash in the crucible is dissolved in 5 ml of HCl (1:2) and is transferred quantitatively into a beaker and heated in the fume hood for 30 minutes below boiling point (below 100°C). If necessary we add more HCl solution so that the volume in the beaker is kept at about 5 ml. After 30 minutes the solution is filtered through the filter paper that has been previously rinsed with HCl solution. The filtrate is transferred into a 100 ml volumetric flask. The beaker is rinsed with some deionized water and the contents are added through the filter paper into the flask. The flask is filled with deionized water to the mark and well stirred. 5 ml of the solution is then pipetted into a 50 ml measuring flask and filled with deionized water to the mark.

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)	g_P2O5(g/L)	m_P2O5(µg)
Blank probe	-	-	160	80	80	80			-	-	-
Calibr.sol.1	-	20	140	80	80	80			0,0403	0.0057	2.288
Calibr.sol.2	-	40	120	80	80	80			0.0806	0.0114	4.576
Calibr.sol.3	-	60	100	80	80	80			0.1209	0.0172	6.864
Calibr.sol.4	-	80	80	80	80	80			0.1612	0.0229	9.152
Calibr.sol.5	-	100	60	80	80	80			0.2015	0.0286	11.44
Calibr.sol.6	-	120	40	80	80	80			0.2418	0.0343	13.728
Calibr.sol.7	-	140	20	80	80	80			0.2821	0.0400	16.016
Calibr.sol.8	-	160	-	80	80	80			0.3224	0.0458	18.304
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)} ·	50 · 100

	0.16 · 5

*w (P₂O₅) =*	m_P2O5

	m_sample

The amount of the phosphates is calculated by the following equation:

w(P₂O₅)_added = w(P₂O₅)_total - b

b = 0.0179 * w(proteins)

0.0179 =	% P₂O₅ (average value in fresh meat)

	% proteins (average value in fresh meat)

w(proteins) = percent of the proteins calculated from the amount of water, fats and ash or read from the declaration of the product

Developed and prepared by: Janja Dvor�ek, Kristina Frlic, Tadeja Polajnar, Biotechnical School Kranj

5. Spectrometric determination of phosphates in meat products