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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.
The total nitrogen content of milk: the nitrogen content, expressed in per cent by mass, as determined by the specified Kjeldahl method.
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.
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.
c (1/2 H2SO4) or c (HC1) = 0,1 mol/1 standardized to the nearest 0,0001 mol/1.
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.
Usual laboratory equipment and, in particular:
Kjeldahl flasks of capacity 500 ml.
Suitable boiling aids, for example, glass beads of diameter approximately 5 mm, Hengar granules, pumice.
Burette or automatic pipette, to deliver 1,0 ml.
Graduated measuring cylinders, glass, of capacities 50, 100 and 250 ml.
Digestion apparatus in an inclined position (approximately 45o), with electric heaters or gas burners that do not heat the flasks above the level of their contents, with a fume extraction system.
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.
Pipette or automatic pipette, to deliver 0,10 ml.
Conical flasks, of capacity 500 ml, graduated at 200 ml.
Burette of capacity 50 ml, gratuated in 0,1 ml, maximum error ± 0,05 ml.
Magnifying lens, for reading the burette (5.9.).
pH meter
Automatic burette.
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.
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.
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.).
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.
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.
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.
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.
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).
Note: The titration of the blank distillate will require only a very small volume of the standard volumetric solution (4.7.).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.).
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.
Calculate the nitrogen content (WN), expressed in grams of nitrogen per 100 g of product by:
where:
=
the nitrogen content.
=
the volume in millilitres of the standard volumetric solution of acid used in the determination.
=
the volume in millilitres of the standard volumetric solution of acid used in the blank test,
=
the concentration, expressed as moles per litre of the acid standard volumetric solution (4.7.).
=
the mass in grams of the test portion.
Round off the result to the nearest 0,001 g per 100 g.
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.