INTERNATIONAL Iso STANDARD 1874 smne00-01 Food and feed products — General guidelines for the determination of nitrogen by the Kjeldahl method Produits alimentaires el aliments des animaux — Lignes directrices (générales pour le dosage de Vazote selon le méitnade de Mjstiahi150 4871:2009() Introduction “The analyss of proucs of animal or plant origin, such 25 those used in food and feed products, often Includes determining thei ritopen content according tote Kjeldahl method “This metnos canbe stansardized in prncpla, a5 itis generly accepted that ciferent ascarsus or operating sretroge are equivalent finer ‘enuts re smi “The purpose of tis document isto descnbe the various stages ofthe method. the associated eral points {nd the minum cojmcivest be schaves fo anaure atthe matnos Spped coveety. “This document provides generat guidlines: ts not ntended to replace exsing ntemabonal Standards whichFood and feed products — General guidelines for the determination of nitrogen by the Kjeldahl method WARNING — The use of this Intemational Standard may involve hazardous materials, operations and ‘equipment. This Intemational Standard does not purport to address all the safety problems associated with its use. Itis the responsibility of the user of this international Standard to establish appropriate ‘safety and heslth practices and determine the applicability of regulatory limitations prior to use. 1 Scope ‘This International Standard provides general guidelines for the determination of nitrogen by the Kjeldahl ‘method. It applies to food and feed products containing nitrogenous compounds tnat can be directly Getarmined by the Kjaldsh) matned NOTE This measurement genepie coes netiase into account me nvegen tam nirates and nities. 2 Principle Digestion of test porion wih concentrated sulfuric acid in the presence of catalysts to convert the organic: rivagan into ammonium sufste Excess sodium hyéroxide is added to the coclad cigest to release the ‘ammonia, The released ammonia is ditlled into an excess of boric acd solution and ten tivated wan = andsrd solution of sufure or hydrochlere acid. The nirogen content is esleulated from the quantity of ‘ammonia produced NOTE intefotgwing txt. ne term ‘trogen refers to organi mtregen 3. Reagents Use only reagents of recognized analytical grade. unless othenvise specified, and distilled or demineralized water or water of equivalent purty 3.1 Sulfurio acid, vinually fee from nitrogenous compounds and of mass density gag = 1.83 giml to 1B4 gin. 2.2. Catalysts (see £21). 3.3. Borie acid solution (10 i to 40gil degending on the apparatus used). If using the colorimetiic fendigoint firaton, Eerie acid solution shall contain indieater (the pH or colour of this moved eoltion shall B= {cused before use) 3.4, Standard hydrochloric acid (0.02moll to OE0 mot") oF sulfurie acid solution (0.01 mot! to 0.28 moll) The tire ofthe solution. ¢, shal be known fot least within 0,00! moll 4.5 Indicators, which should change colour between pH 4 and gH &. NOTE Various intestows are avaliable A metn! rec and ciomocresol teen mixes inaictor le mastcommaniy used ‘Rezo}teuea Done geld eoliions contamng mad ingestore are valaDI227 Sodium hydroxide solution, min 20% mass faction. 38 Antifoaming agents SAMPLE bieoo, mul pate 29 Ammonia sulfate or ammonium chloride (niniraum pusty 08.9%) lovmadistely before use, dey the ammonium suliate or ammonium chloside at 104°C 44 °C for at least 2h. Alo #2 e0i st arnbiant temperature m3 sesionster NOTE Solutions etncum cencsnaton canbe utes 3:10 Tryptophan or acetanilide or lysine hydrochloride (minimum pursy 89 % mass fraction). ‘These rengente should be kept any fram hum WARNING — Do nat ary there reagents in an oven before Use 2:44 Suerose, with ntrogen content laze than 3 mass tration ef0,002 % WARNING — De not dry suerase in an oven before use. 4 Apparatus and materials Usual laboratory apparatus and, in particular. the following 441 Analytical balance, capable of weighing to the nearest 0.001 g 42 Digestion, steam distillation and titration systems. “They are used to perform the operations desorbed in Clause 6 and to ensure thatthe performance cbjectivas scribed in 6.5.3 and 6.5.4 are mat. 43 Bolling point regulators (f needed), for example pumice grains, glass beads, aluminium oxde (corundum) oF silicon earbid. 44 Weighing paper oc medium, tree from nitrogenous compounds and suitable for the test portion and {ype of product 5 Operating method NOTE __Accorsng tothe nature ot ne sample, may Ge neseseary fo pragare ne test portion In agvance to coin a homogeneous cample (grinding, hamogenizalln, cic). 6.1 Testportion ‘The test partion, the quantity of which depends on the presumad nitrogen cantent determined by the Kjeldahl ‘method, shall be represantatve of the sample and eontain between 0,006 g and 0.2 g of niogen. “The test portion ean be cbtainad by weighing witn the analytical balance (4.1) to give mass. min grams or by Using 2 pipette, to ve volume, Tn miles.‘The test portion can be insartad into the tube directly or via'@ suppor (4.4). ‘The quanty of test parton ean be adjusted according to the compostion of the product undar test and the ‘quantiy of sufure acd (sae 6.22). 5.2 Digestion 5.24. Catalysts It is important to differentiate between the substances used to rsige the boling point of the Sauid during Gigeston and the catalysts themseives tat factate digestion. The former are usually potassium sulle of, ‘possibly. soclum sulfate. They are intoduced in suffcient quantity to raise the bong pomnt of the acd 10 between 380 °C and 420°C. The most commonly used catalyst is Copper in the form of copper suste alone oF mated win tkanlum axe, ‘The optional adsiton of hydrogen peroxide (26) on the basis of Si to Smi ger tube grier to heating _scoalaries cgeston, Gut snould be used with tne vimost care to ensure that no nirogen is lost the form of ‘vapour. Moreover, great care should be taken when adding hydrogen peroxide tothe tubes, as this causes @ sttong exothermic reaction The quantiy of potassium suffte provided by the catalyst should not be less than 7g Depending an the sectors of actvty, various compestions are used. They should mast the requirements. of the blank test (5.5.2) and the contol tests (53 and 554) (Speratar should handle selenium-based catalysts and waste conditions wih care NOTE Ressy-o-es composts catayte are avaliable on me manct forexampil nade arpa om). 5.22 Addition of sid It is important to use 2 suffcient quantty of sulfuric aid to ensure digestion after — acid consumption by the organic matter ofthe sample, heaving in mind the fact that 1g of fat consumes 10 ml of suture acd, 1g of protein consumes 5 ml ef sulfuric acid, 1g of earbonycrate consumes 4 milof sulle eid -— seid consumption by the reagents (sats) — eid losses by evaporation. ‘The addition of 20 ml to 25 ml of acd (2.1) is generaly suficent for good digestion and to maintain excess ‘acid at the end ofthe reaction 5.23. Heating WARNING — The following operations should be performed under a very wall ventilated fume hood, ‘The manufacturers instructons relating to tre use of the equipment should generally be followed. The Gigeston system shoula be made homogeneous, for example ty cresting # thermal or digeston affcency process diagram (53). Foam-preducing agents should be brought to baling point by increasing the temperature gradually or in steps. ‘Three to four arops ef antfeaming agent cer tube (22) ean also be Uses‘Acid fumes shal be removed with an extraction system suitable forthe equipment used. Excessive exrction ‘may cause crystalization and a loss of nrogen (see Annex A). In al casas, the eigestion temperature and time should be determined to mest the requirements of the cigeston conoltast (6.6.3), NOTE 1 Keath 21 420°C orto nou approprat fr numerous mato=s “Tha digest obtained should be eles ni tres ftom black parties [At the and of the digestion process, allow the tubas to coo! sway from any possible contamination. At this Stage, the test potions can be stored and dill later NOTE 2 Dhuton step win water 25 descebeain 5.3.1 may be.one at ins Stage oraer 1b avold eytauton. 5.3 Ammonia distillation 52.4. Alkalinization Diute the digest with water, and then alkalis by adding at least 35 ml of sodium hystosie solution (2.7) par rile of sure acid (3.1) used fr the digestion process. NOTE The volume tthe sadasecolmn mydroe eluon 2.7] may be lower mats fecton|e higher than 90% WARNING — Care should be taken when adding the solution, as the medium becomes very hot. 592.2. Distilation PPecforn the ditilation wih the apparatus under consideration ints usval condiion. Collet the distilte in the boric acid solution (3.3) which shall contain the indicator (35). Aust the pH until there is a change of colour ‘tm grey (bromocresol green + methyl red indicator) before beginning the distlation “There are several eters for determining when the distilaton process is fished, for example, when a spectic volume of citilate nas teen colecieg, ste 2 fixed cisblston time, and s0 on. Ensure tht in complince with the conto esis (5), the ammoni citation is complete and that there is no excess by entrainment of the akaline iquia 84. Titration “The distilate obtained is trated with suture (2.1) or hydrochlore acid (24): this ean be done simultaneously or attr cististon, Postaitlston stration snould be pertormad as s000 as possivie afer ditlston. “There are bie methods of cetecting the en pit. — By visual colorimetny oF using an opticel measurement system: The end point is reached when the ‘nestor changes colour In the esse of visual colorimetry, «ss important io fivate each test refereng tO the conditions obtained inthe blank test. — By potentiometric analysis wth 2 pH measurement system: Dapending on the equipment or operating methods. the end point may be 3 fixed pH (ganeraly BH 4.5, which corresponds to the inlecten port of the tration curve), the pH obtained in te Bink test ofthe orginal pH of the bore acid solution Inboth cases, the validity f the thration operations should be checked 2s desenbed in 6.5.4 Facord the volumas of irant obtsinad: Ii forthe blank test and I forthe samles.‘Ore STE Test and St TESST ote AISUIGEST COATGT TERT and One GGEIGN CONT TAHT SHOU Be WOUTEE each set of nizogen determinatian tests 552 Blanktest Perform 2 blank test using the operating method descrbed above, replacing the liquid test porion withthe [same volume of water and sading the appropriate quantly of sucrose (217) NOTE in lane any sgeston combo! tute, we eueote ls utes ae organs mater te coneume 8 quatey ot euture 230i equatentig nator fest partor currg ajeion. 352. Digestion test Farform a cigesion cont test using the operating method descrted stove, repacing the test corion with the same quantity of typicghan or acetanilide or isine hydrochloride (2.10) as the quandty of nitrogen in the [Eumpes and adsing the apprapnsta quantty of crore (217), CCatoulate the percentage mass fracion of nitrogen recovered, which should be between 05 % and 101%, 55.4 Distilationsitration tests Perform a blank distillation-traton test using the operating method described in 5.3, but without atest portion. ‘The volume obtained should be suntractad Wom tnat ofthe dablstontraton est Perfor a ditlation-ttration contol test undar the same conditions an atest portion of ammonium ea (3.8) ‘corresponding tothe quanbty of trogen in the samples. CCaleuiste the pereentape mass fracion of nivogen recovered, which should be between 09% and 101 %, 6 Expression of results ‘The nitrogen content, exsessed a5 2 percentage mass fraction orn grams par 100 mi, i equal to: ,-Ta)ee, «14100 ext 00D WaTy)ee, x14 100 7 xt O00 isthe mass, in grams, ofthe test poston: 41 isthetive, in moles per tre, ofthe hydrochloric 2eid or suture ac Tg isthe volume, in miliies, of hydrochloric set or sulfuric acd used inthe blank test tration: 7, isthe volume, in miles, of hydrochloric aed or sulfuric acd used in te tet ponion tration; F,_ isthe volume, in militares, of the test portionAnnex A (informative) Potential sources of error Faults noted Causes Solutions proposed During digestion [Tao much spray or foam Too rapdaise intemperate Reduce the esing ie or ads the steps Fat or sweet sample tests Use an antiiming agent Too high 2 volume of quid samples | Use a smaller test porion Back panicles in the digest Inappropriate digestion [Optimize the concitons: check the timantemperstre Jsigestion (55.3) Check ine samplelacideataiyst Proportions Pelt enstaication Less of acid due toa too powerful [Redvoe the exvacton rate t can fume exraction system {be reduced as soon as ne white itumes disappes? Check the samplelaciicatalyst Propartons 2. During distillation and nitrogen content determination [Ditlstercivaton estresuk [Less efammens: [Check the apparatus for tghtness 20 low (seals and glass instruments) Insuficien toric aia Increase the concentration or volume of tie Borie acid solution Ammonis entrainmantincompiete|Inevease the catilaton time: ‘neorrzct aod thre measurement |Tarste the a0 Bank cistitation-tiraton test result [Perform a new blank test too han [Disstonciraten estresut [Incorrect acid tire messuremant | irate he acid too high Potion due to ammonia vapaur _|Aveid handing ammonia in the \veiney [Eninment ef sodium hydroxide in |Reduce the volume of water added the cette before sietiston Digestion tex resultioo ow inappropriate digestion (Optimize te concions: check the timetemperstire Jdigeston (55.3) Check ine samplelaciseatayst prosortons