饲料中粗蛋白测定方法.docx

1、饲料中粗蛋白测定方法饲料中粗蛋白测定方法 The determination method of crude protein in feed 1、原理 Principle 凯氏法测定试样中的含氮量，即在催化剂作用下，用硫酸破坏有机物，使含氮物转化成硫酸铵。加入强碱进行蒸馏使氨逸出，用硼酸吸收后，再用酸滴定，测出氮含量，将结果乘以换算系数6.25，计算出粗蛋白含量。Kjeldahl method for nitrogen , destroyed organic with sulfuric acid by catalyst, and made nitrogen change into ammoni

2、um sulfate. To mix with the alkali and distilling with the purpose of ammonia overflowing , with boric acid absorption, then use acid titration, to measure the nitrogen content, multiplied by the coefficient factor of 6.25, the result to calculate the crude protein content. 2、试剂Reagent 2.1硫酸（GB625）：

3、化学纯，含量为98，无氮。Sulfuric acid (GB625) : chemical pure and content is 98%, without nitrogen. 2.2混合催化剂：0.4g硫酸铜，5个结晶水（GB665），6g硫酸钾（HG3920）或硫酸钠（HG3908），均为化学纯，磨碎混匀。Mixed catalyst: 0.4g cooper sulfate , 5 crystallization water (GB665), 6g potassium sulphate (HG3-920) or sodium sulfate (HG3-908) ,above all ar

4、e chemical pure, grinding blending .2.3氢氧化钠（GB629）：化学纯，40水溶液（m/V）。Sodium hydroxide (GB629) : chemical pure , 40% aqueous solution(m/v) 2.4硼酸（GB628）：化学纯，2水溶液（m/V）。Boric acid (GB628): chemical pure , 2% aqueous solution ( m/v) 2.5混合指示剂：甲基红（HG3958）0.1乙醇溶液，溴甲酚绿（HG31220）0.5乙醇溶液，两溶液同等体积混合，在阴凉处保存期为三个月。Mixe

5、d indicator : Methyl red (HG3-958) 0.1 ethanol solution , bromocresol green (HG3-1220) 0.5%ethanol solution, mixed together base on equal volume and the validity date for 3 month in the shade . 2.6盐酸标准溶液：邻苯二甲酸氢钾法标定，按GB601制备。Standard solution of hydrochloric acid:potassium hydrogen phthalate method o

6、f calibration ,according to GB601.2.6.1盐酸标准溶液：c（HCl）=0.1mol/L。8.3mL盐酸（GB622，分析纯），注入1000mL蒸馏水中。Standard solution of hydrochloric acid: c（HCl）=0.1mol/L . 8.3ml hydrochloric acid( GB 622, analytical pure) , with 1, 000 ml of distilled water .2.6.2盐酸标准溶液：c（HCl）=0.02mol/L。1.67mL盐酸（GB622，分析纯），注入1000mL蒸馏水中

7、。Standard solution of hydrochloric acid: c（HCl）=0.02mol/L. 1.67ml hydrochloric acid( GB 622, analytical pure) , with 1, 000 ml of distilled water . 2.7蔗糖（HG31001）：分析纯。Cane sugar (HG3-1001) : analytical pure .2.8硫酸铵（GB1396）：分析纯，干燥。Ammonium sulfate (GB1396) : analytical pure , dry . 2.9硼酸含量吸收液：1硼酸水溶液1

8、000mL，加入0.1溴甲酚绿乙醇溶液10mL，0.1甲基红乙醇溶液7mL，4氢氧化钠水溶液0.5mL，混合，置阴凉处保存期为一个月（全自动程序用）。Absorption liquid of boric acid : 1000ml boric acid aqueous solution of 1% boric acid , add into 10 ml bromocresol green aqueous solution of 0.1% , and 7ml methyl red ethanol solution of 0.1% , 0.5ml sodium hydroxide aqueous

9、solution of 4% , mixed together and validity date for 1 month in the shade . （Atomical control ) 3、仪器设备 Instrument 3.1实验室用的样品粉碎机或研钵。The sample pulverizer or mortar for laboratory use3.2分样筛：孔径0.45mm（40目）。Sample sieve: aperture 0.45mm (40 mesh) 3.3分析天平：感量0.0001g。Analytical balance : sensitive quality

10、0.0001g3.4消煮炉或电炉。Digestion furnace or electric stove3.5滴定管：酸式，10、25mL。Burette :acid , 10,25ml 3.6凯氏烧瓶：250mL。Kjeldahl flask :250ml3.7凯氏蒸馏装置：半微量水蒸气蒸馏式。Kjeldahl distillation equipment : Semimicro water vapor distillation type3.8锥形瓶：150、250mL。Conical flask :150,250ml 3.9容量瓶：100mL。Volumetric flask :100ml

11、 3.10消煮管：250mL。Digestion tube :250ml 3.11定氮仪：以凯氏原理制造的各类型半自动。Azotometer : manufacturing various types of semi-automatic in light of kjeldahl principle4、试样的选取和制备 The selection & preparation of specimen 选取具有代表性的试样用四分法缩减至200g，粉碎后全部通过40目筛，装于密封容器中，防止试样成分的变化。To select the representative specimen and reduce

12、 to 200g by quartering , after pulverized all through the 40mesh, packed in the sealed container, to prevent changes in specimen composition.The constant Kjeldahl determination digestion and distillation equipment1- Hydraulic extraction pipe; 2-Tap ; 3-Inversion of the drying tube; 4-Kjeldahl flask;

13、 5& 7 -electric stove ; 8-distillation flask; 6&9 -iron lining ; 10 -sampling hooper ; 11-Condenser pipe ; 12 -receiving flask 5分析步骤 Analytical procedure 5.1.1试样的消煮 The specimen digestion称取试样0.51g（含氮量580mg）准确至0.0002g，放入凯氏烧瓶中，加入6.4g混合催化剂，与试样混合均匀，再加入12mL硫酸和2粒玻璃珠，将凯氏烧瓶置于电炉上加热，开始小火，待样品焦化，泡沫消失后，再加强火力（360

14、410）直至呈透明的蓝绿色，然后再继续加热，至少2h。Take 0.5 1g specimen (nitrogen content of 5 80 mg) accurate to 0.0002 g in the kjeldahl flask, mixed with 6.4 g of catalyst, and mix the specimen , and add 12 ml of sulphuric acid and 2 grain of glass beads, placed kjeldahl flask in the heating furnace with soft fire, stay

15、 specimen coking, after the foam disappeared, to strengthen the fire (360 410 ) until the specimen change into translucent turquoise, and then continue heating for at least 2 h.5.1.2氨的蒸馏：The nitrogen distillation 将试样消煮液冷却，加入20mL蒸馏水，转入100mL容量瓶中，冷却后用水稀释至刻度，摇匀，做为试样分解液。将半微量蒸馏装置的冷凝管末端浸入装有20mL硼酸吸收液和2滴混合指示

16、剂的锥形瓶内。蒸汽发生器的水中应加入甲基红指示剂数滴，硫酸数滴，在蒸馏过程中保持此液为橙红色，否则需补加硫酸。准确移取试样分解液1020mL注入蒸馏装置的反应室中，用少量蒸馏水冲洗进样入口，塞好入口玻璃塞，再加10mL氢氧化钠溶液，小心提起玻璃塞使之流入反应室，将玻璃塞塞好，且在入口处加水密封，防止漏气。蒸馏4min降下锥形瓶使冷凝管末端离开吸收液面，再蒸馏1min，用蒸馏水冲洗冷凝管末端，洗液均流入锥形瓶内，然后停止蒸馏。Make the boiled liquid of specimen cooling,and add 20 ml of distilled water into 100 m

17、l of volumetric flask, after cooling diluted to scale with water, shake up,as the decomposition liquid of specimen . The half trace distiller condenser pipe tip into the conical flask which equipped with 20 ml of boric acid absorbing liquid and 2 drops of mixed indicator .Steam generator should add

18、few drops of methyl red indicator , few drops of sulfuric acid to keep the liquid for the orange red during the distillation process , otherwise need to add the sulfuric acid. Accurately moving 10 20 ml sample decomposition fluid injection in the reaction chamber of distillation unit, and rinse the

19、inlet with few of distilled water, cork the inlet glass stopper , add 10 ml solution of sodium hydroxide, carefully lift glass stopper into the reaction chamber, and add water seal at the entrance, to prevent air leakage.Distillation for 4 min lowered conical flask from condenser pipe end to absorb

20、liquid level, distillation again for 1 min, washed the condenser pipe end with distilled water, lotion into inside the conical flask, and then stop the distillation.5.1.2.3蒸馏步骤的检验 The distillation step test精确称取0.2g硫酸铵，代替试样，按5.1.2步骤进行操作，测得硫酸铵含氮量为21.190.2，否则应检查加碱、蒸馏和滴定各步骤是否正确。Accurately weigh 0.2g of

21、ammonium sulfate, instead of specimen , and operate according to 5.1.2 steps, measured the nitrogen content of ammonium sulfate was 21.19 + / - 0.2% , otherwise should check the steps of alkaline, distillation and titration, wether it is correct .5.1.3滴定 Titration 用5.1.2.1或5.1.2.2法蒸馏后的吸收液立即用0.1mol/L

22、或0.02mol/L（4.6.2）盐酸标准溶液滴定，溶液由蓝绿色变成灰红色为终点。After distillation the absorption liquid immediately titration with 0.1 mol/L or 0.02 mol/L (4.6.2) of hydrochloric acid standard solution by 5.1.2.1 or 5.1.2.2 method , the solution of ash from blue-green to red for the finish.6、空白测定Blank determination 称取蔗糖0

23、.5g，代替试样，按第5章进行空白测定，消耗0.1mol/L盐酸标准溶液的体积不得超过0.2mL。消耗0.02mol/L盐酸标准溶液体积不得超过0.3mL。Weighting 0.5g of sucrose, instead of specimen , the blank determination according to chapter 5, consumption of 0.1 mol/L hydrochloric acid standard solution volume should not exceed 0.2 mL. Consumption of 0.02 mol/L hydro

24、chloric acid standard solution volume should not exceed 0.3 mL. 7、分析结果的表述 The description of analysis result 7.1计算见下式：To calculate as below : 粗蛋白质（）=（V2-V1）c0.01406.25/（mV/V）100the crude protein(%)= （V2-V1）c0.01406.25/（mV/V）100式中：V2滴定试样时所需标准酸溶液体积，mL； The volume of required standard acid solution whe

25、n specimen titration , ml ; V1滴定空白时所需标准酸溶液体积，mL； The volume of required standard acid solution when blank titration , ml ; c盐酸标准溶液浓度，mol/L； The concentration of hydrochloric acid standard solution, mol/L ; m试样质量，g； The specimen quality , g V试样分解液总体积，mL； The total volume of decomposition liquid, ml ;

26、 V试样分解液蒸馏用体积，mL； The distillation volume of decomposition liquid, ml ; 0.0140与1.00mL盐酸标准溶液c（HCl）=1.000mol/L相当的、以克表示的氮的质量。 Equal to 1.00 mL of hydrochloric acid standard solution (c (HCl) = 1.000 mol/L) , the quality of nitrogenexpressed in grams .6.25氮换算成蛋白质的平均系数。 Average coefficient of nitrogen con

27、version into proteins7.2重复性 Repeatability 每个试样取两个平行样进行测定，以其算术平均值为结果。 Each specimen taken for determination of two parallel samples, its arithmetic mean value as result.当粗蛋白质含量在25以上时，允许相对偏差为1。 When the crude protein content more than 25% , allows the relative deviation is 1%.当粗蛋白含量在1025之间时，允许相对偏差为2。 When the crude protein content within 10%25% , allows the relative deviation is 2%.当粗蛋白质含量在10以下时，允许相对偏差为3。 When the crude protein content less than 10% , allows the relative deviation is 3%.