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Commission Regulation (EC) No 152/2009 of 27 January 2009 laying down the methods of sampling and analysis for the official control of feed (Text with EEA relevance)
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This method makes it possible to determine the crude protein content of feed on the basis of the nitrogen content, determined according to the Kjeldahl method.
The sample is digested by sulphuric acid in the presence of a catalyst. The acid solution is made alkaline with sodium hydroxide solution. The ammonia is distilled and collected in a measured quantity of sulphuric acid, the excess of which is titrated with a standard solution of sodium hydroxide.
Alternatively, the liberated ammonia is distilled into an excess of boric acid solution, followed by titration with hydrochloric acid or sulphuric acid solution.
When colorimetric end-point detection is applied, methyl red and bromocresol indicators must be added to the boric acid solutions. If 1 litre of the boric acid solution is prepared, before adjusting to volume, 7 ml methyl red indicator solution (3.16) and 10 ml bromocresol green solution (3.17) shall be added.
Dependent on the water used, the pH of the boric acid solution might differ from batch to batch. Often an adjustment with a small volume of alkali is necessary to obtain a positive blank.
:
The addition of about 3 ml to 4 ml of NaOH (3.11) into 1 litre of 10 g/l boric acid usually gives good adjustments. Store the solution at room temperature and protect the solution from light and sources of ammonia fumes during storage.
Other concentrations of volumetric solutions (3.5, 3.6, 3.7, 3.10, 3.11, and 3.19) can be used, if this is corrected for in the calculations. The concentrations shall always be expressed to four decimal places.
Apparatus suitable for performing digestion, distillation and titration according to the Kjeldahl procedure.
Weigh 1 g of the sample to the nearest 0,001 g and transfer the sample to the flask of the digestion apparatus. Add 15 g of potassium sulphate (3.1), an appropriate quantity of catalyst (3.2) (0,3 to 0,4 g of copper (II) oxide or 0,9 to 1,2 g of copper (II) sulphate pentahydrate), 25 ml of sulphuric acid (3.4) and if required, a few granules of pumice stone (3.12) and mix.
Heat the flask moderately at first, swirling from time to time if necessary until the mass has carbonised and the foam has disappeared; then heat more intensively until the liquid is boiling steadily. Heating is adequate if the boiling acid condenses on the wall of the flask. Prevent the sides from becoming overheated and organic particles from sticking to them.
When the solution becomes clear and light green continue to boil for another two hours, then leave to cool.
Add carefully enough water to ensure complete dissolution of the sulphates. Allow to cool and then add a few granules of zinc (3.3), if required. Proceed according to 5.2.1 or 5.2.2.
Place in the collecting flask of the distillation apparatus an exactly measured quantity of 25 ml of sulphuric acid (3.5) or (3.7) depending on the presumed nitrogen content. Add a few drops of methyl red indicator (3.8).
Connect the digestion flask to the condenser of the distillation apparatus and immerse the end of the condenser in the liquid contained in the collecting flask to a depth of at least 1 cm (see observation 8.3). Slowly pour 100 ml of sodium hydroxide solution (3.9) into the digestion flask without loss of ammonia (see observation 8.1). Heat the flask until the ammonia has distilled over.
Where titration of the ammonia content of the distillate is performed manually, the procedure mentioned below applies. Where the distillation unit is fully automated to include titration of the ammonia content of the distillate, follow the manufacturer's instructions for operation of the distillation unit.
Place a collecting flask containing 25 ml to 30 ml of the boric acid solution (3.18) under the outlet of the condenser in such a way that the delivery tube is below the surface of the excess boric acid solution. Adjust the distillation unit to dispense 50 ml of sodium hydroxide solution (3.9). Operate the distillation unit in accordance with the manufacturer's instructions and distil off the ammonia liberated by the addition of the sodium hydroxide solution. Collect distillate in the boric acid receiving solution. The amount of distillate (time of steam distillation) depends on the amount of nitrogen in the sample. Follow the instructions of the manufacturer.
In a semi-automatic distillation unit, the addition of excess sodium hydroxide and the steam distillation are performed automatically.
Proceed according to 5.3.1 or 5.3.2.
Titrate the excess sulphuric acid in the collecting flask with sodium hydroxide solution (3.10 or 3.11) depending on the concentration of the sulphuric acid used, until the end-point is reached.
Titrate the contents of the collecting flask with the hydrochloric acid standard volumetric solution (3.19) or with the sulphuric acid standard volumetric solution (3.6) using a burette and read the amount of titrant used.
When colorimetric end-point detection is applied, the end-point is reached at the first trace of pink colour in the contents. Estimate the burette reading to the nearest 0,05 ml. An illuminated magnetic stirrer plate or a photometric detector may aid visualisation of the end-point.
This can be done automatically using a steam distiller with automatic titration.
Follow the manufacturers' instructions for operation of the specific distiller or distiller/titrator.
:
When an automatic titration system is used, titration begins immediately after distillation starts and the 1 % boric acid solution (3.18) is used.
Where a fully automatic distillation unit is employed, the automatic titration of the ammonia can also be carried out with end-point detection using a potentiometric pH system.
In this case an automatic titrator, with a pH-meter is used. The pH-meter shall be calibrated properly in the range of pH 4 to pH 7 following normal laboratory pH-calibration procedures.
The pH end-point of the titration is reached at pH 4,6, being the steepest point in the titration curve (inflection point).
To confirm that the reagents are free from nitrogen, carry out a blank test (digestion, distillation and titration) using 1 g of sucrose (3.14) in place of the sample.
Calculations are performed according to 6.1 or 6.2.
The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:
where:
=
is the volume (ml) of NaOH (3.10 or 3.11) used in the blank test,
=
is the volume (ml) of NaOH (3.10 or 3.11) used in the sample titration,
=
is the concentration (mol/l) of sodium hydroxide (3.10 or 3.11),
=
is the weight (g) of sample.
The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:
where:
=
is the weight (g) of the test portion,
=
is the concentration (mol/l) of the standard volumetric solution of the hydrochloric acid (3.19),
=
is the volume (in ml) of hydrochloric acid used for the blank test,
=
is the volume (in ml) of hydrochloric acid used for the test portion.
The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:
where:
=
is the weight (g) of the test portion,
=
is the concentration (mol/l) of the standard volumetric solution of sulphuric acid (3.6),
=
is the volume (in ml) of sulphuric acid (3.6) used for the blank test,
=
is the volume (in ml) of sulphuric acid (3.6) used for test portion.
The difference between the results of two parallel determinations carried out on the same sample must not exceed:
0,2 % in absolute value, for crude protein contents of less than 20 %,
1,0 % relative to the higher value, for crude protein contents from 20 % to 40 %,
0,4 % in absolute value, for crude protein contents of more than 40 %.
Carry out the analysis (digestion, distillation and titration) on 1,5 to 2,0 g of acetanilide (3.13) in the presence of 1 g of sucrose (3.14); 1 g acetanilide consumes 14,8 ml of sulphuric acid (3.5). Recovery must be at least 99 %.
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