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) (c. 152)

ANNEX III U.K. METHODS OF ANALYSIS TO CONTROL THE COMPOSITION OF FEED MATERIALS AND COMPOUND FEED

C. DETERMINATION OF THE CONTENT OF CRUDE PROTEIN U.K.

1. Purpose and scope U.K.

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.

2. Principle U.K.

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.

3. Reagents U.K.

3.1. Potassium sulphate. U.K.

3.2. Catalyst: copper (II) oxide CuO or copper (II) sulphate pentahydrate, CuSO₄ 5H₂O. U.K.

3.3. Granulated zinc. U.K.

3.4. Sulphuric acid, ρ20 = 1,84 g/ml. U.K.

3.5. Sulphuric acid, standard volumetric solution, c(H₂SO₄) = 0,25 mol/l. U.K.

3.6. Sulphuric acid, standard volumetric solution, c(H₂SO₄) = 0,1 mol/l. U.K.

3.7. Sulphuric acid, standard volumetric solution, c(H₂SO₄) = 0,05 mol/l. U.K.

3.8. Methyl red indicator; dissolve 300 mg of methyl red in 100 ml of ethanol, σ = 95 %-96 % (v/v). U.K.

3.9. Sodium hydroxide solution (Technical grade may be used) β = 40 g/100 ml (m/v: 40 %). U.K.

3.10. Sodium hydroxide, standard volumetric solution c(NaOH) = 0,25 mol/l. U.K.

3.11. Sodium hydroxide, standard volumetric solution c(NaOH) = 0,1 mol/l. U.K.

3.12. Granulated pumice stone, washed in hydrochloric acid and ignited. U.K.

3.13. Acetanilide (m.p. = 114 ^oC, N-content = 10,36 %). U.K.

3.14. Sucrose (nitrogen free). U.K.

3.15. Boric acid (H₃BO₃). U.K.

3.16. Methyl red indicator solution: dissolve 100 mg methyl red in 100 ml ethanol or methanol. U.K.

3.17. Bromocresol green solution: dissolve 100 mg bromocresol green in 100 ml ethanol or methanol. U.K.

3.18. Boric acid solution (10 g/l to 40 g/l depending on the apparatus used). U.K.

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.

Note

:

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.

3.19. Hydrochloric acid standard volumetric solution c(HCl) = 0,1 mol/l. U.K.

Note:

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.

4. Apparatus U.K.

Apparatus suitable for performing digestion, distillation and titration according to the Kjeldahl procedure.

5. Procedure U.K.

5.1. Digestion U.K.

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.

5.2. Distillation U.K.

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.

5.2.1. Distillation into sulphuric acid U.K.

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.

5.2.2. Distillation into boric acid U.K.

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.

Note:

In a semi-automatic distillation unit, the addition of excess sodium hydroxide and the steam distillation are performed automatically.

5.3. Titration U.K.

Proceed according to 5.3.1 or 5.3.2.

5.3.1. Sulphuric acid U.K.

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.

5.3.2. Boric acid U.K.

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.

Note

:

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).

5.4. Blank test U.K.

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.

6. Calculation of results U.K.

Calculations are performed according to 6.1 or 6.2.

6.1. Calculation for titration according to 5.3.1 U.K.

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

where:

V_o

=

is the volume (ml) of NaOH (3.10 or 3.11) used in the blank test,

V₁

=

is the volume (ml) of NaOH (3.10 or 3.11) used in the sample titration,

c

=

is the concentration (mol/l) of sodium hydroxide (3.10 or 3.11),

m

=

is the weight (g) of sample.

6.2. Calculation for titration according to 5.3.2 U.K.

6.2.1. Titration with hydrochloric acid U.K.

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

where:

m

=

is the weight (g) of the test portion,

c

=

is the concentration (mol/l) of the standard volumetric solution of the hydrochloric acid (3.19),

V₀

=

is the volume (in ml) of hydrochloric acid used for the blank test,

V₁

=

is the volume (in ml) of hydrochloric acid used for the test portion.

6.2.2. Titration with sulphuric acid U.K.

The content of crude protein, expressed as a percentage by weight, is calculated according to the following formula:

where:

m

=

is the weight (g) of the test portion,

c

=

is the concentration (mol/l) of the standard volumetric solution of sulphuric acid (3.6),

V₀

=

is the volume (in ml) of sulphuric acid (3.6) used for the blank test,

V₁

=

is the volume (in ml) of sulphuric acid (3.6) used for test portion.

7. Verification of the method U.K.

7.1. Repeatability U.K.

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 %.

7.2. Accuracy U.K.

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 %.

8. Observations U.K.

8.1. Apparatus may be of the manual, semi-automatic or automatic type. If the apparatus requires transference between the digestion and distillation steps, this transfer must be carried out without loss. If the flask of the distillation apparatus is not fitted with a dropping funnel, add the sodium hydroxide immediately before connecting the flask to the condenser, pouring the liquid slowly down the side. U.K.

8.2. If the digest solidifies, recommence the determination using a larger amount of sulphuric acid (3.4) than that specified above. U.K.

8.3. For products with a low nitrogen content, the volume of sulphuric acid (3.7) to be placed in the collecting flask may be reduced, if necessary, to 10 or 15 ml and made up to 25 ml with water. U.K.

8.4. For routine analysis, alternative methods of analysis can be applied for the determination of crude protein but the Kjeldahl method described in this Part C is the reference method. The equivalence of the results obtained with the alternative method (e.g. DUMAS) compared to the reference method must be demonstrated for each matrix individually. As the results obtained with an alternative method, even after having verified the equivalency, might deviate slightly from the results obtained with the reference method, it is necessary to mention in the analytical report the method of analysis used for the determination of crude protein. U.K.