1、BRITISH STANDARDAnimal feeding stuffs Determination of nitrogen content and calculation of crude protein content Part 1: Kjeldahl methodICS 65.120g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3
2、g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO5983-1:2005Incorporating corrigendum September 2008BS EN ISO 5983-1:2005National forewordThis British Standard is the UK implementation of EN ISO 5983-1:2005. It is identical with ISO 5983-1:2005, incorporating corrigendum September 2008. It sup
3、ersedes BS 5766-4:1998 which is withdrawn.The start and finish of text introduced or altered by corrigendum is indicated in the text by tags. Text altered by ISO corrigendum September 2008 is indicated in the text byThe UK participation in its preparation was entrusted to Technical Committee AW/10,
4、Animal feeding stuffs.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.Compliance with a British Standard cannot
5、 confer immunity from legal obligations.This British Standard was published under the authorityof the Standard Policy and Strategy Committee on 23 August 2005 BSI 2009 ISBN 978 0 580 65653 8Date Comments Implementation of ISO corrigendum September 2008. AC1AC1Amendments/corrigenda issued since publi
6、cation30 April 2009 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 5983-1July 2005ICS 65.120English VersionAnimal feeding stuffs - Determination of nitrogen content andcalculation of crude protein content - Part 1: Kjeldahl method(ISO 5983-1:2005)Aliments des animaux - Dtermination de la tene
7、ur enazote et calcul de la teneur en protines brutes - Partie 1:Mthode Kjeldahl (ISO 5983-1:2005)Futtermittel - Bestimmung des Stickstoffgehaltes undBerechnung des Rohproteingahaltes - Teil 1: Kjeldahl-Verfahren (ISO 5983-1:2005)This European Standard was approved by CEN on 27 June 2005.CEN members
8、are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to
9、the Central Secretariat or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same stat
10、us as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,Slovenia, S
11、pain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2005 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Me
12、mbers.Ref. No. EN ISO 5983-1:2005: EForeword This document (EN ISO 5983-1:2005) has been prepared by Technical Committee ISO/TC 34 “Agricultural food products“ in collaboration with Technical Committee CEN/TC 327 “Animal feeding stuffs - Methods of sampling and analysis“, the secretariat of which is
13、 held by NEN.This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by January 2006, and conflicting national standards shall be withdrawn at the latest by January 2006. According to the CEN/CENELEC Interna
14、l Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherla
15、nds, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Endorsement notice The text of ISO 5983-1:2005 has been approved by CEN as EN ISO 5983-1:2005 without any modifications. BS EN ISO 5983-1:2005iiiContents Page1 Scope . 12 Normative references . 13 Princ
16、iple. 14 Reagents and materials . 15 Apparatus 26 Sampling 27 Preparation of test sample. 38 Procedure 39 Calculation and expression of results 510 Precision 611 Test report . 7Annex A (informative) Results of interlaboratory test 8Bibliography . 10BS EN ISO 5983-1:20051Animal feeding stuffs Determi
17、nation of nitrogen content and calculation of crude protein content Part 1: Kjeldahl method 1 Scope This part of ISO 5983 specifies a method for the determination of the nitrogen content of animal feeding stuffs by the Kjeldahl process, and a method for the calculation of the crude protein content.
18、The method does not measure oxidized forms of nitrogen or heterocyclic nitrogen compounds. This method does not distinguish between protein nitrogen and non-protein nitrogen. If it is important to determine the content of non-protein nitrogen, an appropriate method should be used. 2 Normative refere
19、nces The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 6498, Animal feeding stuffs Preparation
20、of test samples 3 Principle The organic matter is digested by sulfuric acid in the presence of a catalyst. The reaction product is rendered alkaline, then the liberated ammonia is distilled and titrated. The nitrogen content is calculated and the result is multiplied by the conventional factor to ob
21、tain the crude protein content. 4 Reagents and materials Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or deionized water or water of equivalent purity. The reagents except the standard materials (4.6) shall be practically free from nitrogenous compounds
22、. 4.1 Potassium sulfate.4.2 Catalyst, either 4.2.1 or 4.2.2. 4.2.1 Copper(II) oxide (CuO). 4.2.2 Copper(II) sulfate pentahydrate (CuSO45H2O). 4.3 Sulfuric acid, c(H2SO4) 18 mol/l, U20(H2SO4) 1,84 g/ml. BS EN ISO 5983-1:200524.4 Paraffin wax.4.5 Saccharose.4.6 Standard materials, either 4.6.1 or 4.6.
23、2. 4.6.1 Acetanilide, with melting point 114 C; nitrogen (N) content 103,6 g/kg. 4.6.2 Tryptophan, with melting point 282 C; nitrogen (N) content 137,2 g/kg. Dry before use. 4.7 Sodium hydroxide solution, w(NaOH) 33 % (mass fraction). 4.8 Collecting liquid, either 4.8.1 or 4.8.2. 4.8.1 Sulfuric acid
24、, standard volumetric solution, c(H2SO4) 0,05 mol/l or c(H2SO4) 0,125 mol/l. 4.8.2 Boric acid, U(H3BO3) 40 g/l. 4.9 Solutions for titration.4.9.1 Sodium hydroxide, standard volumetric solution, c(NaOH) 0,1 mol/l or c(NaOH) 0,25 mol/l. 4.9.2 Sulfuric acid, standard volumetric solution, c(H2SO4) 0,05
25、mol/l or c(H2SO4) 0,125 mol/l. The molarity of standard volumetric solutions should be known to the fourth decimal point. 4.10 Mixed indicator, neutral point at pH 4,4 to 5,8. Dissolve 2 g of methyl red and 1 g of methylene blue in 1 000 ml of ethanol M(C2H5OH) 95 % (volume fraction). 4.11 pH indica
26、tor paper.4.12 Boiling aids, such as granulated pumice stone, or glass beads of diameter 5 mm to 7 mm, or carborundum chips, washed in hydrochloric acid and in distilled water, and ashed. 5 ApparatusUsual laboratory apparatus and, in particular, the following. 5.1 Analytical balance.5.2 Digestion, d
27、istillation and titration apparatus.6 SamplingA representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling is not part of the method specified in this part of ISO 5893. A recommended sampling method isgiven in ISO 6
28、497. Store the sample in such a way that deterioration and change in its composition are prevented. BS EN ISO 5983-1:200537 Preparation of test sample Prepare the test sample in accordance with ISO 6498. 8 ProcedureWARNING The operations described in 8.3.1 and 8.3.2 should be carried out under a wel
29、l-ventilated hood or in a fume cupboard which is resistant to sulfuric acid. 8.1 GeneralFor general directions on the application of the Kjeldahl method, see ISO 1871. 8.2 Test portion Weigh, to the nearest 1 mg, a mass of the test sample chosen according to the expected nitrogen content so that the
30、 test portion contains between 0,005 g and 0,2 g of nitrogen and, preferably, more than 0,02 g. The mass of the test portion of homogeneous air-dry samples should be between 0,5 g and 2,0 g. The mass of the test portion of wet and/or inhomogeneous samples should be between 2,5 g and 5,0 g. 8.3 Deter
31、mination8.3.1 Digestion of organic matter Transfer the test portion quantitatively into a Kjeldahl digestion flask of suitable size (usually 800 ml). Add 15 g of potassium sulfate (4.1). Add an appropriate quantity of catalyst as follows: 0,3 g of copper(II) oxide (4.2.1) or 0,9 g to 1,2 g of copper
32、(II) sulfate pentahydrate (4.2.2). Add 25 ml of sulfuric acid (4.3) for the first gram of dry matter of the test portion and 6 ml to 12 ml for each additional gram of dry matter. Mix thoroughly, ensuring complete wetting of the test portion. Support the flask so that its axis is inclined at an angle
33、 of 30 to 45 to the vertical. Maintain the flask in this position throughout heating. Heat the flask moderately at first to prevent foam from rising into the neck of the flask or escaping from the flask. NOTE 1 It may be advisable to add an anti-foaming agent such as paraffin wax (4.4). Heat moderat
34、ely, swirling from time to time, until the mass has carbonized and the foam has disappeared. Then heat more intensively until the liquid is boiling steadily. NOTE 2 Heating is adequate if the boiling acid condenses towards the middle of the neck of the Kjeldahl flask. Avoid overheating of the walls
35、of the flask not in contact with liquid. NOTE 3 If a naked flame is used, such overheating can be prevented by placing the flask on a sheet of heat-resistant material with an aperture of diameter slightly less than that of the flask at the liquid level. After the liquid has become clear with a light
36、 green-blue colour, heat for another 2 h. Leave to cool. If the digest starts to solidify, add some water and mix by swirling. BS EN ISO 5983-1:200548.3.2 Distillation of ammonia 8.3.2.1 Carefully add 250 ml to 350 ml of water to dissolve the sulfates completely. If necessary, facilitate dissolving
37、by heating the flask in warm water. Mix by swirling and allow to cool.Add a few boiling aids (4.12). For some specific samples, the sulfates may not completely dissolve in the added water. In that case, it is recommended to repeat the digestion with a reduced mass of potassium sulfate (4.1). 8.3.2.2
38、 Pipette, into the collecting flask of the distillation apparatus, 25 ml of the sulfuric acid (4.8.1), choosing the concentration according to the expected nitrogen content of the test portion. Add 100 ml to 150 ml of water. Add a few drops of the mixed indicator (4.10). Proceed in accordance with 8
39、.3.2.4. 8.3.2.3 Alternatively, transfer into the collecting flask 100 ml to 250 ml of boric acid (4.8.2). Add a few drops of mixed indicator (4.10). Simultaneous titration of the ammonia (see 8.3.3.3) during distillation is recommended since it facilitates verification of the end of distillation. 8.
40、3.2.4 Immerse the end of the condenser in the liquid contained in the collecting flask, to a depth of at least 1 cm. Slowly pour 100 ml of sodium hydroxide solution (4.7) into the digestion flask along the wall. Immediately connect the flask to the distillation apparatus. Heat the flask in such a ma
41、nner that approximately 150 ml of distillate is collected in 30 min. At the end of this time, check the pH of the distillate at the tip of the condenser using litmus paper (4.11). If the reaction is alkaline, continue distillation. IMPORTANT Lift the condenser from the liquid just before the end of
42、the distillation, to prevent backflow. If, during distillation using sulfuric acid as collecting liquid, the contents of the collecting flask become alkaline, recommence the determination, making appropriate adjustments. 8.3.3 Titration 8.3.3.1 Titration with automatic endpoint indication using a pH
43、-meter is recommended. Otherwise, the endpoint is indicated by the change in colour of the mixed indicator (4.10) added in 8.3.2. 8.3.3.2 If sulfuric acid is used as the collecting liquid, titrate, in the collecting flask, the excess sulfuric acid with sodium hydroxide solution (4.9.1), c(NaOH) 0,1
44、mol/l or c(NaOH) 0,25 mol/l as appropriate, until the endpoint is indicated by the pH-meter or until the colour changes from violet to green. 8.3.3.3 If boric acid is used as the collecting liquid, titrate the ammonia with sulfuric acid (4.9.2), c(H2SO4) 0,05 mol/l or c(H2SO4) 0,125 mol/l as appropr
45、iate, until the endpoint is indicated by the pH-meter or the colour changes from green to violet. If simultaneous titration is not possible (see 8.3.2.3), the titration should be carried out as soon as possible after the distillation is complete, ensuring that the temperature of the distillate does
46、not exceed 25 C. Under these conditions, losses of ammonia are avoided. 8.4 Blank test Perform a blank test using about 1 g of saccharose (4.5) in place of the test portion. BS EN ISO 5983-1:200558.5 Check test Perform a check test by determining the nitrogen content of acetanilide (4.6.1) or trypto
47、phan (4.6.2) after addition of 1 g of saccharose (4.5). The choice of the substance for the check test should be related to the digestibility of the samples to be analysed. Acetanilide is easily digested, whereas the digestion of tryptophan is more difficult. The recovery of nitrogen from acetanilid
48、e or tryptophan should be at least 99,5 % for acetanilide and at least 99,0 % for tryptophan. 9 Calculation and expression of results 9.1 Calculation of nitrogen content 9.1.1 Distillate collected in sulfuric acid Provided that the volumes of sulfuric acid used to collect the ammonia for the determi
49、nation (8.3) and for the blank test (8.4) are equal, calculate the nitrogen content, wn1, in grams per kilogram of the test sample, by the following equation: 01 1n1VV cMwmuu where V0is the volume, in millilitres, of the sodium hydroxide solution (4.9.1) required for the blank test; V1is the volume, in millilitres, of the sodium hydroxide solution (4.9.1) required for the determination; c1is the concentration, in moles per litre, of the sodium hydroxide solution (4.9.1) used for the titrations; M is the molar mass, i
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