Council Decision of 14 November 1992 laying down methods for the analysis and testing of heat-treated milk for direct human consumption (92/608/EEC)

IV. DETERMINATION OF TOTAL NITROGEN CONTENT U.K.

1. SCOPE AND FIELD OF APPLICATION U.K.

This procedure specifies the reference method for the determination of the total nitrogen content of raw milk and of whole milk, partly skimmed milk and skimmed milk.

2. DEFINITION U.K.

The total nitrogen content of milk: the nitrogen content, expressed in per cent by mass, as determined by the specified Kjeldahl method.

3. PRINCIPLE U.K.

A weighed quantity of the milk sample is digested with concentrated sulphuric acid and potassium sulphate and copper (II) sulphate as catalyst, in order to convert the nitrogen of the organic compounds into ammonium sulphate. The ammonia is released by the addition of sodium hydroxide solution and then distilled and absorbed in a boric acid solution. This is titrated with an acid solution.

4. REAGENTS U.K.

4.1. Potassium sulphate (K₂SO₄). U.K.

4.2. Copper sulphate solution. Dissolve 5,0 g of copper (II) sulphate pentahydrate (CuSO₄, 5H₂O) in water and dilute to 100 ml (at 20 ^oC) in a volumetric flask. U.K.

4.3. Sulphuric acid, at least 98,0% (m/m) H₂SO₄. U.K.

4.4. Sodium hydroxide solution, 47% (m/m) 704 g NaOH/1 (20 ^oC). U.K.

Note: A less concentrated sodium hydroxide solution may be used for example: 40% (m/m) 572 g/1, 20 ^oC; or 30% (m/m) 399 g/1, 20 ^oC. U.K.

4.5. Boric acid solution. Dissolve 40 g of boric acid (H₃BO₃) in one litre of hot water, allow to cool, and store in a borosilicate glass bottle. U.K.

4.6. Indicator solution. Dissolve 0,01 g methyl red, 0,02 g bromothymol blue and 0,06 g bromocresol green in 100 ml of ethanol. Store the solution in a brown closed bottle, in a cool, dark place. U.K.

4.7. Volumetric solution U.K.

c (¹/₂ H₂SO₄) or c (HC1) = 0,1 mol/1 standardized to the nearest 0,0001 mol/1.

4.8. Nitrogen-free sucrose. U.K.

4.9. Ammonium salt, pure, such as ammonium oxalate (NH₄)₂C₂O₄,H₂O or ammonium sulfate (NH₄)₂SO₄. U.K.

4.10. Tryptophan (C₁₁H₁₂N₂O₂), phenacetin (C₁₀H₇CH₂CONH₂) or lysine mono- or di-hydrochloride (C₆H₁₄N₂O₂ HCl or C₆H₁₄N₂O₂ · 2HCl). U.K.

Note: The purity of reagents in 4.9. and 4.10. should be of higher quality than ‘analytical grade’. If available, certified ammonium salt solution (4.9.) should be used. U.K.

5. APPARATUS AND GLASSWARE U.K.

Usual laboratory equipment and, in particular:

5.1.

Kjeldahl flasks of capacity 500 ml.

5.2.

Suitable boiling aids, for example, glass beads of diameter approximately 5 mm, Hengar granules, pumice.

5.3

Burette or automatic pipette, to deliver 1,0 ml.

5.4.

Graduated measuring cylinders, glass, of capacities 50, 100 and 250 ml.

5.5.

Digestion apparatus in an inclined position (approximately 45^o), with electric heaters or gas burners that do not heat the flasks above the level of their contents, with a fume extraction system.

5.6.

Distillation apparatus, made of borosilicate glass, to which a Kjeldahl flask (5.1.) can be fitted, consisting of an efficient splash-head connected to an efficient condenser with straight inner tube and an outlet tube attached to its lower end; the connecting tubing and stopper (s) shall be close-fitting and preferably of neoprene rubber.

5.7.

Pipette or automatic pipette, to deliver 0,10 ml.

5.8.

Conical flasks, of capacity 500 ml, graduated at 200 ml.

5.9.

Burette of capacity 50 ml, gratuated in 0,1 ml, maximum error ± 0,05 ml.

5.10.

Magnifying lens, for reading the burette (5.9.).

5.11.

pH meter

5.12.

Automatic burette.

6. PROCEDURE U.K.

6.1. To the Kjeldahl flask (5.1.) add boiling aid (5.2.) (eg. three glass beads), 15 g of potassium sulphate (4.1.), 1,0 ml of copper sulphate solution (4.2.), approximately 5 g of milk sample (weighed to the nearest 0,001 g) and 25 ml of sulphuric acid (4.3.). Use the acid to wash down any copper sulphate solution, potassium sulphate or milk on the neck of the flask, and gently mix the contents of the flask. U.K.

Note: Because organic matter consumes sulphuric acid during boiling, use 30 ml of H₂SO₄ (4.3.), instead of 25 ml for digestion, if the milk contains more than 5,0% (m/m) of fat. This should also be done in the blank test. U.K.

6.2. Heat each Kjeldahl flask on the digestion apparatus (5.5.), very gently at first so that all the black froth stays within the bulb. When the initial frothing has ceased and copious white vapour appears, boil vigorously (acid vapour will condense half-way up the neck of the flask) until no black particles remain and until the digest becomes clear pale blue-green in colour. Then boil gently for at least 1,5 hours. Note the following requirements: U.K.

(a)

The time for the digest to become clear should not be more than one hour, and the total digestion time shall not be less than 2,5 hours. If more than one hour is necessary to achieve clearing, the total digestion time shall be increased accordingly.

(b)

The added potassium sulphate promotes the digestion as it raises the boiling temperature of the mixture. If the residual volume of H₂SO₄ is less than approximately 15 ml at the end of the digestion time, nitrogen may have been lost because of excessive heating. If heating by gas, heat the flask on a plate of heat-insulating material, provided with a circular opening of such a diameter that the free flame only touches the part of the flask that is below the surface of the liquid contents (5.5.).

(c)

If black particles enter the neck of the flask and are not all washed down into the bulb by the acid refluxing during the inital stages of the vigorous boiling period (this may be facilitated by rotating the flask) allow the flask to cool sufficiently and carefully wash with the minimum of water. Then continue the digestion as described above.

6.3. When the Kjeldahl flasks are cool, add 300 ml of water (see note) to each so as to wash carefully down the neck of the flask, and mix the contents thoroughly ensuring that the crystals which have separated out are dissolved. Add some boiling aid (5.2.) to ensure uniform boiling. Then to each flask, add 70 ml of sodium hydroxide solution (4.4.) (see note) by gently pouring the solution down the inclined neck of the flask to form a bottom layer in the bulb; do not wet the top of the neck with the sodium hydroxide solution. U.K.

Note: It is necessary that the combined volume of water and sodium hydroxide solution total 370 ml to enable approximately 150 ml of distillate to be collected just before irregular boiling (‘bumping’) ensues (6.4.). Thus, if a larger equivalent volume of a sodium hydroxide solution which is less concentrated than 47 % (m/m) is added, the volume of water added shall be reduced accordingly. For example, if 85 ml of 40 % (m/m) or 125 ml of 30% (m/m) sodium hydroxide solution are to be added, the volume of water added shall be 285 ml or 245 ml respectively. U.K.

6.4. Immediately connect each Kjeldahl flask to a distillation apparatus (5.6.). Ensure that the tip of the condenser outlet-tube is immersed in 50 ml of boric acid solution (4.5.) together with 0,20 ml (5 - 6 drops) of indicator solution (4.6.) all contained in a conical flask (5.8.). Swirl the contents of each Kjeldahl flask to mix thoroughly and boil, but gently at first to prevent excessive frothing. When 100 to 125 ml of distillate have been collected, lower each conical flask until the tip of the condenser outlet-tube is approximately 40 mm above the 200 ml mark. Continue each distillation until irregular boiling (‘bumping’) starts and then immediately stop the heating. Disconnect each Kjeldahl flask and rinse the tip of each condenser outlet-tube with a little water, collecting the rinsings in the conical flask. Note the following requirements: U.K.

(a)

The distillation rate shall be such that approximately 150 ml of distillate are collected when irregular boiling (‘bumping’) starts, the volume of the contents of each conical flask will then be approximately 200 ml.

(b)

The efficiency of each condenser should be such that the temperature of the contents of each conical flask does not exceed 25 ^oC during the distillation.

6.5 Titrate each distillate with standard volumetric solution (4.7.) until the pH is 4,6 ± 0,1, using a pH meter and if desired an automatic burette. Addition of an indicator helps to check whether the titration is proceeding correctly. Take each burette reading to the nearest 0.01 ml with the aid of a magnifying lens (5.10.) avoiding errors of parallax. U.K.

The titrating may be carried out with the indicator only. Titrate until the colour of the distillate corresponds to that of a solution recently prepared from 150 ml of water to which has been added 50 ml of the boric acid solution and 0,20 ml of the indicator contained in a conical flask (5.8).

6.6. Carry out a blank test according to 6.1. to 6.5. inclusive, taking 5 ml of distilled water together with about 0,1 g of sucrose (4.8.) through the procedure instead of the milk sample. U.K.

Note: The titration of the blank distillate will require only a very small volume of the standard volumetric solution (4.7.). U.K.

6.7. Regularly check the accuracy of the procedure by using two recovery trials following the procedure according to 6.1. to 6.5. inclusive. U.K.

6.7.1. Check that no loss of nitrogen occurs as a result of excessive heat or mechanical leaks during distillation, by using a test portion of 0,15 g of ammonium oxalate or sulphate (4.9.) weighed to the nearest 0,001 g together with 0,1 g of sucrose (4.8.). U.K.

The percentage of nitrogen recovered shall be between 99,0 and 100,0 %.

Lower or higher results will indicate failures in the procedure and/or inaccurate concentration of the standard solution (4.7.).

6.7.2. Check that the digestion procedure is sufficient to release all the protein nitrogen by using a test portion of 0,20 g of pure tryptophan, 0,35 g of phenacetin or 0,20 g of lysine hydrochloride (4.10.). All weighings should be to the nearest 0,001 g. At least 98—99% of the nitrogen should be recovered. U.K.

7. SAFETY PRECAUTIONS U.K.

When working with concentrated sulphuric acid and sodium hydroxide and when handling Kjeldahl flasks, always wear a laboratory coat, safety goggles and acid resistant gloves.

During distillation, never leave Kjeldahl flasks unattended. Because of potential danger, stop distillation immediately if flask contents ‘bump’ too vigorously. If the power goes off for more than two to three minutes, lower the collecting flask so that the distillation tip is out of the liquid.

8. EXPRESSION OF RESULTS U.K.

8.1. Calculation and formula: U.K.

Calculate the nitrogen content (W_N), expressed in grams of nitrogen per 100 g of product by:

where:

W_N

=

the nitrogen content.

V

=

the volume in millilitres of the standard volumetric solution of acid used in the determination.

V_o

=

the volume in millilitres of the standard volumetric solution of acid used in the blank test,

c

=

the concentration, expressed as moles per litre of the acid standard volumetric solution (4.7.).

m

=

the mass in grams of the test portion.

Round off the result to the nearest 0,001 g per 100 g.

8.2. Precision U.K.

8.2.1. Repeatability (r): 0,007 g per 100 g. U.K.

8.2.2. Reproducibility (R): 0,015 g per 100 g. U.K.

9. MODIFIED PROCEDURES U.K.

9.1. Use a block digestion apparatus fitted with cylindrical flasks, instead of the digestion apparatus and the Kjeldahl flasks described in 5.5. and 5.1. In this case to identify potential trouble, each spot has to be checked individually (6.7.). U.K.

9.2. Use of steam distillation instead of direct heating of the flasks (6.4.). When the apparatus does not allow the use of distilled water, care should be taken that the water does not contain acid or alkaline volatiles. U.K.

9.3. A test portion of 1 g of milk (semi-macro Kjeldahl) can be used instead of 5 g (6.1.) provided: U.K.

• the amounts of the reagents used for mineralization (6.1.): H₂SO₄, CuSO₄ · 5 H₂O, K₂SO₄, are reduced to the same ratio (1/5).

• the total digestion time (6.2.) is reduced to 75 minutes.

• the amount of sodium hydroxide solution (6.3.) is reduced to the same ratio (1/5).

• an acid standard solution (4.7.) of lower concentration (0,02 to 0,03 mol/l) has to be used.

Note: Using one or more of these options is acceptable only if the repeatability value (8.2.1.) and the two accuracy tests results (6.7.) are in accordance with the requirements given in this method. U.K.