‘Anat FEED Chapter 4, p. 34 4.2.14 AOAC Official Method 2001.11 Forage (Plant Tissue), Grain, and Oilseeds Block Digestion Method Using Copper Catalyst ‘and Steam Distillation into Boric Acid First Action 2001 Final Action 2005 [Applicable to the determination of 0.5-S0% Kjeldahl N 3— 300% equivalent crude protein) in forage, animal feed and pet food, ‘grain, and oilseeds, and applicable to the same matrixes as 976.05 (see 4.2.05), 976.06 (see 4.2.06), 984.13 (see 4.2.09), 988.05 (see 4.2.03), and 990.02 (see 4.2.07); the method does not measure oxidized forms of N or heterocyclic N compounds.) ‘See Tables 2001.11A and B for results ofthe interlaboratory study, expressed on a protein bat is (N * 6.25), supporting acceptance of the method. A. Principle ‘The material is digested in H,SO, to convert the protein N 10 (NH),SO, ata boiting point elevated by the addition of K,SO, with 2 Cu catalyst to enhance the reaction rate. Ammonia is liberated by alkaline steam distillation and quantified titrimetrcally with standardized acid, Aluminum block heaters inerease the efficiency of the digestion. The digest must contain residual H,SO, to retain the NH, Water is added manually or automatically to the digest to avoid mixing concentrated alkali with concentrated acid and to prevent the digest from solidifying. Concentrated NaOH is added to neutralize the acid and make the digest basic, and the liberated NH, is distilled into a boric acid solution and titrated with a stronger standardized acid, HCL, to a colorimetric endpoint. The same endpoint detection system (e.g, indicator, wave length) must be used for the standardization of the HCI and forthe analyte. ‘The analyte is referred to as “crude” protein because the method. determines N, a component of all proteins. In addition, N’from sources other than true protein is also determined. (Additional AOAG OFFICIAL METHODS OF ANALYSIS 265 the chemicals. Do not mix concentrated acid and NaOH dicey. tf ‘chemicals are splashed on the skin or in the eyes, flush with copious B. Apparatus 18) Digestion block—Aluminum alloy block with adjustable ees fr mcasng ndcorrolingbloktempentae {CTeeator Digestion System 20, 1015 Digestor, Foss North Ameren, Eden Prairie, MN, USA; Tel: +1-952-974-9892, Fax: +1-952.975. 19823, info@fossnorthamerica.com; or equivalent, (b) Digestion tubes.—250 ml. (©) Disillation units. (1) For steam distillation. —Foss Tecator 2200, or equivalent, to accept 250 mL digestion tubes and 500 mL. titration flasks. (2) For steam distillation and autotitration —Foss ‘Tecator 2300, of equivalent. (@) Turation flask—S00 mL. graduated Erlenmeyer flask (fr collection and titration of distillate) (©) Fume exhaust manifold —With Tefton ring seals, connected to.a water aspirator in a hooded sink. (O Weighing paper—Low N, Alfie Packers No. 201 (Alfie Packers, Inc, Omaha, NE, USA), of Fisher 09-898-12A, 3 * 3 in, (96% 76 rom), or equivalent. (@) Pipetting dispenser: 5 pint (24 L) acid bottle, ©. Reagents (2) Sulfuric acid —Concentrated, 95-98% 1,80, reagent grade @) Catelyst—7.0 g K,SO, + 08 g CuSO, (Commercially available in tablet form as 3.5 g K,SO, and 04g CuSO, per table.) (©) Sodium hydroxide solution—40% (wiv) NaOH, low N (Sng Ni). 25 mL, adjustable volume, attached 10 Table 2001.11A. Interlaboratory study results for the determination of crude protein by block digestion with a copper catalyst and distillation into 4% boric acid iD No.of fabs" Mean, % RSD, % RSD, % HorRat Protein Block 10(1) 40.19 0.45 076 0.383 ‘Swine petets 1001) 37.04 oar 0.60 0.286 Com silage " 7.10 168 2.16 0.728 Grass hay " ma 1.94 1.94 0.680 Fish meal " 6467 073 0.98 0.460 Dog food " 24.50 087 ot 0.369 Chinchilla food " 18.01 0.89 099 0383 ‘Aun 1011) 79.14 0.40 04a 021 Birdseed " 13.48 0.88 1.29 0475 Meat and bone meal " 50.06 1.00 1.90 08s? ‘Mik replacer " 2078 1.39 430 0.550 Soy boans 9@ 38.76 049 oss 0.236 Sunflower seeds 1" 17.43 2.38 maa 0916 Legume hay 1 18.81 1.45 ae 0.565 7 Each values he aber of abort tained ai elinaon Of OU Sh vale Peitowos Ws Tab Soop TOSIOT SS (©2019 AAG INTERNATIONALAOAC OFFICIAL METHODS OF ANALYSIS (2010) Table 2001.18. Interlaboratory study rosults for the recover copper catalyst and distillation inte boric aeich ANIMAL Feo Chapter 4, p. 35 ry of nitrogen from standard compounds by block digestion with a Gompound| No. oflabs' Theoretical yiald, % N ‘Avg. found, %N Avg. rec. % RSD, % HorRat Aoetaniié 10(0) 10.36 10.37 ‘00.1 1180 053) Lysine HCl 10(0) 158.34 13.92 858 416 4.53 Tryptophan 1010) 13.72 13.55 98.8 1.04 0.39 "Each valve isthe umber of laboratories retained far eiminalion of elles; each value in paresis fs he numer of aboralrss removed outers (@ Metly ved indicator soluion. Dissolve 100 mg methyl red in 100 mL. methanol. (6) Bromocresol green indicator solution— ‘bromocresol green in 100 mL methanol (9 Borie acid solution—4% (wh). Dissolve 400 g HBO, in 5-6 L hot deionized water. Mix and add more hot deionized water to volume of about 9 L, Cool to room temperature, add 100 mL. ‘bromocresol green solution and 70 mL methyl red solution, and dilute toa final votume of 10 L. Adjust to obtain a positive blank of 0,05-0.15 mL with 30 ml. H,BO, solution, using 0.1 M NaOH (to inrease blank) oF 0.1 M HCI (to decrease blank). Commercially available. (€) Borie acid solution.—1% (wi). (Optional trapping solution for tirators that automatically begin titration when disilation begins.) Dissolve 100 g H,BO, in 5-6 L hot deionized water, mi and add more hot deionized water to 2 volume of about 9 L.. Cool to room temperature, add 100 mL bromocresol green solution and 70 mL methyl red solution, and dilute to & final volume of 10 L. Commercially availabe. (t) Hsdrochloric acid standard solution—0.1000 M. Prepare 4s in 936.15 (see A.1.06) or use premade solution of certified specification range 0.0995-0.1005 M, and use 0.1000 M for calculation. Commercially available. () Reference standards —Aramonium sulfite, tryptophan, lysine HCl, or glycine p-toluenesulfonic acid, for use as standard; 99.9%. () Sucrose —N-free. D. Proparation of Analytical Sample issolve 100 mg Grind dry laboratory sample to fineness of grind (ca 0.7-1 mm), which gives a relative standard deviation (RSD) of £2.0% for 10 successive determinations of N in ground mixture of com grain and soybeans (2 + 1). Fineness required to achieve this precision must be used for all dry mixed feeds and other nonuniform materials. Mix liquids to uniformity. E Determination (a) Digestion —Turn on block digestor and heat to 420°C: Weigh material, as indicated below, recording each test portion Weight (W) to the nearest mg for weights of 21 g, and to the est 0.1 mg for weights of “1,0 g. Do not exceed 1.2 g For materials with 3-25% protein, weigh approximately 1.0 tes portion; with 25-50% protein, approximately 0.5 tes portion; and 50% Protsin, approximately 0.3 test po ; () Dry feed, forage, cereal, grain, vilseed. We n is i portion of ground, wellamixed test portion onto a tured low N weighing paper Fold paper around material and drop into & urbered Kjeldahl tube eee, (2) Ligue —Wigh sigh >1 st pation of wlnined ‘aly sample into a smal ‘are beaker. Quantitative ans o a numbered Kjeldahl tube with <20 mL deionized water. Altematively, weigh slightly >1 g well-mixed test portion into 4 small tared beaker. Transfer to a numbered Kjeldahl tube and reweigh beaker. The differential weight loss corresponds to the amount of test portion actually transferred tothe tube. (6) Standards.—Perform quality control analysis and analyses of standards with each batch. The standards available from Hach Co. (Loveland, CO, USA; Tel: +1-800-227-4224 or +1-970-669- 3050), Sigma (St. Louis, MO, USA), J. Baker (Phillipsburg, NJ, USA), the National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA) are listed in Table 2001.11C. The various ammonium salts and glycine p-toluenesulfonate serve primarily as a check on distillation efficiency and accuracy in titration steps because they are digested very readily. Lysine and nicotinic acid p-toluenesulfonate serve asa check on digestion efficiency because they are difficult to digest. Include a reagent blank tube containing a folded low N weighing ‘paper with each batch. (©) Digestion—Ada two catalyst tablets to each tube. Add 12 mL H,SO, to each tube, using pipetting dispenser; add 15 mL {or high fat materials (>10% fat). Mixtures may be held overnight at this point. If mixture foams, slowly add 3 mL. 30-35% H,O,. Let reaction subside in perchloric acid fume hood or in exhaust system. Table 2001.11C. Standards ‘Approximate Thooretical Standard woight.g yield, % N. ‘Ammonium p-toluenesulfonate 05 7402 (Hach 2279-24), Glycine p-toluenesuifonate 08 5.665 (Hach 22780-24), [Nicotinic acid p-toluenesulfonate 0.2 4743 (Hach 2781-24) Lysine monohydrochloride on 15.34 (Sigma .-6626) ‘Acetanilide (Bakor AO68-05) 03 10.38 ‘Tryptophan (Sigma T 8659) 02 13:72 ‘Ammonium salts Diammoniam hydrogen phosphate 0.2 221 (100% assay) ‘Ammonium chloride (100% assay) 0.2 26.18 ‘Ammonium sulfate (100% assay) 02 212 ‘Ammonium dihydrogen phosphate 0.3 12.18 (NIST 200) Cittus leaves (NIST 1572) 10 2868 Urea (NIST 2141) on 46.63‘Animtat FEED Chapter 4, p. 36 ‘Attaca heat side shields to tube rack. Place fume manifold tightly ‘on tubes, and turn water aspirator on completely. Place rack of tbes in proheated block. After 10 min, tum water aspirator down until ‘acid fumes are just contained within exhaust hood. A condensation zone should be maintained within the tubes. After bulk of sulfur ide fumes are produced during initial singes of digestion, reduce ‘vacuum source to prevent loss of H,SO , Digest additional 50 min, Total digestion time is approximately 60 min, “Tum digestor off, Remove rack of tubes with exhaust still in place, and put in the stand to cool for 10-20 min. Cooling ean be increased by using commercial air blower o by placing in hood ‘vith bood sash pulled down to increase airflow across tubes. When fuming has stopped, remove manifold, and shut off aspirator. Remove side shields. Let tubes cool. Wearing gloves and eye protection, predilute digests manually before distilling. Carefully ‘add a few milliliters of deionized water to each tube. If spattering ‘occurs, the tubes are too hot. Let cool fora few more minutes. Add water 1 each tube to a fotal volume of approximately 80 mL (liquid level should be about half way between the two shelves of the tube rach). This is a convenient stopping point. If digest solidifies, place tube containing diluted digest in block digester, and carefully warm with occasional swirling until salts dissolve. If distilling unit equipped with steam addition for equilibration is used, the manual dilution steps can be omitted. ‘About 70 mL deionized water is then automatically added during the distillation eyele. (©) Distillarion.—Place 40% NaOH in alkali tank of distillation unit. Adjust volume dispensed to SO mL. Attach digestion tube containing diluted digest to distillation unit, or use automatic dilution feature, if available, Place graduated 500 mL Erlenmeyer titration flask containing 30 mL H,BO, solution with indicator fon receiving platform, and immerse tube from condenser below surface of H,BO, solution. (When an automatic titration system is used that begins titration immediately after distillation start, 19% H,BO, may be substituted.) Steam distil until>150 mL distillate is collected (2180 mL total volume). Remove receiving flask. Titrate H,BO, receiving solution with standard 0.1000 M HCI to violet ccadpoint (just before the solution goes back to pink). Lighted stir plate may aid visualization of endpoint, Record milliliters of HC] toatIeast the nearest 0.05 mL. ‘This is done automatically by using a steam distiller with automatic titration, Follow the manufacturer's instructions for ‘operation of the specific distiller or distllerftitrator. F. Verification of Nitrogen Recovery Run N recoveries to check accuracy of procedure and equipment, (a) Nitrogen loss.—Use 0.12 g (NHi,),S0, and 0.67 g sucrose per flask, Add all other reagents as in E, and distill under same conditions as in E. Recoveries must be 299%, (b) Distillation and titration efficiency—Distill 0.12. @ (NH),SO, omitting digestion, Recoveries must be 299.5%. (©) Digestion efficiency—Use 03 g acetanilide or 0.18 tryptophan, with 0.67 g sucrose per flask, Add all other reagents as stated in E, Digest and distill under same conditions as used for a ‘determination, Recoveries must be 298%, 6. Cafeutations N,)oMe18.01 Kjeldahl nitrogen ge~ Ye) *M214.01 a Wad Cade protein, 6-% Kjeldahl No ©2019 AOAC INTERNATIONAL AOAC OFFICIAL METHODS OF ANALYSIS (2549) “shere V, = volume (mL) of standardized acid used to tice ~ volume (mL) of standardized acid used t0 titrate reagey test; Vi F siandard HCl; 14.01 ~ atomic weight of y, blank; M = molarity of Cvciet () of test portion or standart; 10 ~ ctr to cons and F = factor to convert N to protein w i to percent T factor are 5.70 for wheat, 638 for duty produets, and other feed materials. Reference: J. AOAC Int. 85, 3092002) 4.3.01 AOAC Official Method 941.04 Urea and Ammoniacal Nitrogen in Ani Urease Method First Action 1941 Final Action 2002 A, Reagents (8) Defoaming solution —Dow Coming Corp. Antifoam B Emulsion. (b) Urease solution. Prepare fresh solution by dissolving standardized urease in H,O so that each 10 mL neutralized solution will convert N of 20.1 g pure urea. ‘Standardization. —o determine alkalinity of commercial uesse preparation, dissolve 0.1 g in $0 mL H,O and titrate with 0.1 M HCI, using methyl red, 984,13B(¢) (see 4.2.09). Add same volume 0.1 N HCI to each 0.1 g urease in preparing urease solution. To determine enzyme activity, prepare ca 50 ml neutralized 1% solution, Add different amounts of solution to 0.1 g pure urea and follow with enzyme digestion and distillation as in determination. Calculate activity of urease preparation from amount ofthis wesse solution that completely converted urea, as determined by compete recovery of N by di (€) Caleiu chloride solution—Dissolve 25 g anhydrous CeCl, in 100 mL HO. B. Determination Place 2 g test portion in Kjeldahl flask with ca 250 mL HO. Add 10 ml. urease solution, stopper tightly, and let stand 1st ‘oo temperate o€ 20 min a 40°C. Cool to room temperate necessary, Use additional urease solution if feed contains >5% we (12% protein equivalent). Rinse stopper aed neck with H,0. Add 22 g MgO (heavy type), 5 mL CaCl, solution, and 3 defoaming solution, and connect flask with condenser by Kieks coonsctng bulb, Distill 100 ml. into measured volume tts ‘id, 936.15 (see A.1.06) oF 890.01 (see A1.14), and tte Standard alkali, 936,16 (see 4.1.12), sing methyl red, 9841380) (ee 42.09, References: JAOAC 24, 867(1941); 28, 874(1942);27, 494194