First Commission Directive of 13 November 1979 laying down Community methods of analysis for testing certain partly or wholly dehydrated preserved milk for human consumption (79/1067/EEC) (c. 1067)

ANNEX II U.K. METHODS OF ANALYSIS RELATING TO THE COMPOSITION OF CERTAIN PARTLY OR WHOLLY DEHYDRATED PRESERVED MILK PRODUCTS INTENDED FOR HUMAN CONSUMPTION

METHOD 7: DETERMINATION OF PHOSPHATASE ACTIVITY (MODIFIED SANDERS AND SAGER PROCEDURE) U.K.

4. REAGENTS U.K.

4.1. Solution A U.K.

Barium borate hydroxide buffer: pH 10,6 ± 0,1 at 20 ^o C.

Dissolve: 25,0 g of barium hydroxide (Ba(OH)2.8H2O) in water and dilute to 500 ml.

Dissolve: 11,0 g of boric acid (H3BO3) in water and dilute to 500 ml.

Warm the two solutions to 50 ^o C and mix.

Shake and cool the mixture to room temperature.

Adjust the pH to 10,6 ± 0,1 with the barium hydroxide solution and filter.

Store the solution in a tightly stoppered container.

Before use, dilute the buffer with an equal quantity of water.

4.2. Solution B: U.K.

Colour development buffer.

Dissolve: 6,0 g of sodium metaborate (NaBO2) (or 12,6 g of NaBO2.4H2O) and 20,0 g of sodium chloride (NaCl) in water and dilute to 1 000 ml with water.

4.3. Solution C U.K.

Buffer substrate solution.

4.3.1. Dissolve 0,5 g of disodiumphenylphosphate (Na2C6H5PO4.2H2O) in 4,5 ml of Solution B (4.2). Add 2 drops of Solution E (4.5) and allow to stand 30 minutes. Extract the colour with 2,5 ml butanol (4.10). If necessary, repeat the colour extraction. After separation, discard the butanol. This solution can be kept for several days in a refrigerator. Develop and extract the colour once more before use. U.K.

4.3.2. Pipette 1 ml of this solution into a 100 ml volumetric flask and make up to volume with Solution A. Prepare the buffer solution immediately before use. U.K.

4.4. Solution D U.K.

Precipitant.

Dissolve 3,0 g of zinc sulphate (ZnSO4.7H2O) and 0,6 g of copper (II) sulphate (CUSO4.5H2O) in water and make up to 100 ml with water.

4.5. Solution E U.K.

Gibb's reagent.

Dissolve 0,040 g of 2,6-dibromoquinone 1,4 — chloroimide (O.C6H2Br2.NCl) in 10 ml of 96 % ethanol. Store the solution in a dark glass bottle kept in a refrigerator. Discard this reagent when it has become discoloured.

4.6. Colour dilution buffer U.K.

Dilute 10 ml of Solution B (4.2), colour development buffer, to 100 ml with water.

4.7. Copper sulphate solution U.K.

Dissolve 0,05 g of copper (II) sulphate (CUSO4.5H2O) in water and make to 100 ml with water.

4.8. Phenol standard solution U.K.

Dissolve 0,2 ± 0,001 g of pure phenol in water and make up to 100 ml in a volumetric flask with water. This solution can be stored for several months in a refrigerator. Dilute 10 ml of this solution to 100 ml with water. This diluted solution contains 200 μg of phenol in 1 ml and can be used for preparing more dilute solutions.

4.9. Boiled distilled water. U.K.

4.10. n-Butanol. U.K.