With the rapid development of the feed industry, the prices of feed ingredients and feed products have also remained high. The detection of crude protein is one of the important indicators for evaluating feed ingredients and their products. At present, the Kjeldahl method is commonly used, namely, the “Method for Determination of Crude Protein in Feed†(GB 1996-6432). However, this method also has the disadvantages of a relatively complicated and time-consuming measurement process. In addition to the strict procedures in accordance with the prescribed procedures, certain experimental techniques and practical experience are also required. Therefore, many feed companies always have various problems in the actual operation process, resulting in abnormal test results and I do not know how to analyze. This article takes GB/T 6432-1994 as the standard and deals with the details that should be taken into consideration when determining the standard of Kjeldahl nitrogen determination results.
Explore. Of course, there are relatively simple methods for determining the crude protein content. Considering the complexity of using the national standard method to determine crude protein, and sometimes we do not need to make a very accurate determination of the content of crude protein, we can consider using crude protein determination. Instrument or ZDDN-II Kjeldahl nitrogen analyzer to determine the content of crude protein in the material. Although this kind of instrument lacks the preparation of the national standard method, it is simple and convenient, and the more important reason is that the data it measures can achieve our desired purpose.
1 Determination of Crude Protein: Preparation of Reagents
Chemicals used in the determination of crude protein, such as concentrated sulfuric acid, hydrochloric acid, sodium hydroxide, boric acid, copper sulfate, potassium sulfate (sodium sulfate), ammonium sulfate, and sucrose, are all chemically pure reagents, and the anhydrous carbonic acid used for the calibration of hydrochloric acid standard solution is used. Sodium is the reference reagent. Before the preparation of reagents, be sure to use p H test paper or acidity meter to check whether the distilled water is neutral. The used beakers, reagent bottles and other liquid equipment are cleaned.
1.1 Preparation of hydrochloric acid standard solution
The prepared hydrochloric acid standard solution should be as low as possible, generally C(HCl) = 0.02 to 0.05 mol • L-1. Although the use of low concentrations, but can reduce the operating errors and reading errors. The preparation of hydrochloric acid standard solution must be carried out in strict accordance with the standard operation. The standard anhydrous sodium carbonate is fixed at 270 to 300°C and burned to a constant weight. Weigh it to 0.0001g. Make 4 to 6 parallel samples and remove the highest and lowest values. After averaging, a blank test is also required. The amount of hydrochloric acid standard solution preparation should not be too much, the use of time is too long, to prevent evaporation of water and hydrochloric acid and affect the accuracy of its concentration.
1.2 Preparation of other reagents
400g • L-1 sodium hydroxide solution, 20g • L-1 boric acid solution, mixed indicator (1g • L -1 methyl red ethanol solution and 5g • L -1 bromocresol green ethanol solution equal volume mixing) Weighing should be done quickly, accurately, and steadily, and then it is too long to prevent it from being used. In particular, mixed indicators should not exceed 3 months.
2 Determination of Crude Protein: Selection and Preparation of Samples
2.1 Sampling
The volume and quality of feed ingredients and products are often very large, so the collected samples must be representative, otherwise, even if a series of analytical work is very precise and accurate, its significance is not significant, and sometimes it may even lead to wrong conclusions. Therefore, the correct sampling method should be used. Samples should be taken from several representative areas and then thoroughly mixed into representative samples for the entire feed, and then a small portion of them should be used as analytical samples. In the sampling process, it should be done immediately and objectively to avoid the influence of man-made and subjective factors. It should also be noted that the sample collected must have a certain amount. The amount of the amount collected should be determined as appropriate, depending on the feed ingredients and product moisture content, particle size, uniformity, and number of replicates.
2.2 Sample
The collected samples are often more, and the samples used during the test are often less, so the collected samples should be reduced. In most feed companies and testing facilities, a convenient and simple quartile method is generally used. Conditional units can be sampled using a sampler.
2.3 Powder Sample
The sample of feed is generally granular, so the sample to be crushed is to be crushed. The equipment used for crushing is generally a coffee grinder or a plant sample crusher. After crushing the sample, the crusher generally passes a 40-mesh sieve, and the crushing sample and the remaining parts of the crusher must be thoroughly mixed and thoroughly mixed to avoid crushing. The grades affect the accuracy of the analysis results. For some non-crushable roughage such as straw slag, etc. will remain in the grinder very little through the sieve hole, this part must not be lost, should try to crush and evenly mixed into the sample to avoid causing analysis errors.
2.4 Samples
The amount of the sample is specified in the standard 0.5 ~ 1g, in order to ensure the accuracy of the analysis results, in the analysis process should be based on the level of the sample protein content to determine the weight of the sample, such as concentrates, fish meal, corn protein powder Samples of high protein, such as soybean meal and soybean meal, can be controlled between 0.5 and 1g. Samples of low protein and coarse feed, such as corn, straw, and mash, can be appropriately increased to about 2 to 3g to increase the accuracy of the measurement results. It should also be borne in mind that, when weighing, try to use an electronic analytical balance that is accurate to 0.0001g and placed in Kjeldahl flasks or digestive tubes without loss.
3 Determination of crude protein: digestion
3.1 Catalysts and their usage
Under the premise of small environmental pollution, low price, and good catalytic effect, the current catalysts are mostly copper sulfate and anhydrous potassium sulfate (or anhydrous sodium sulfate). The addition of different companies varies greatly, among which copper sulphate: anhydrous potassium sulphate (or anhydrous sodium sulphate) is mainly 1:9, 1:10, 1:15 and 1:17, and the national standard is 0.4g of copper sulphate and 6g anhydrous potassium sulfate (or anhydrous sodium sulfate) in a ratio of 1:12. If the added amount is increased, the digestible liquid is easy to be agglomerated and difficult to transfer; the amount of addition is reduced, the digestion time is prolonged or the digestion is incomplete. It is recommended that you perform according to the national standard. According to their own experience and the number of samples, ease of digestion, the amount of protein content to adjust.
3.2 The amount of sulfuric acid
The amount of concentrated sulfuric acid should be appropriately adjusted depending on the weight of the sample, the water content, and the level of protein contained in the sample. For large, light and heavy roughage, high protein feeds, or samples with high water content and concentrated starch content, the amount of concentrated sulfuric acid should be increased to about 15 mL. This is because such samples, concentrated sulfuric acid in its dehydration and carbonation consumption is increased, when the amount of concentrated sulfuric acid added is insufficient or just enough sample dehydration and carbonization will appear when the dry phenomenon. Otherwise, you need to add about 10mL. If the ventilation volume of the fume hood is relatively large, add about 2 mL of concentrated sulphuric acid to avoid the evaporation of concentrated sulphuric acid and reduce its concentration. The incomplete digestion of the sample will result in a low measured value.
3.3 Digestion temperature
At the beginning, it was heated at a low temperature (200-300°C) until the coking foam disappeared, gradually increasing the temperature (360-410°C). Initially, the low temperature heating was used to prevent the sample from foaming, and the particles that came out of the flask or after carbonization adhered to the wall of the flask, resulting in incomplete digestion and low measured results. For samples that are rich in fat or are easily foamed during digestion, they can be digested
Before adding a small amount of defoamer, such as octanol, liquid paraffin and so on.
3.4 Digestion time
Digestion time is also a problem that many literatures have been discussing. There are also many studies on rapid digestion. The standard for digestion time is that the digestion is transparent blue-green, and then heated to digest for 15 minutes. According to studies by the Soviet Army, it is advisable to digest the digestate for 30 min after digestion. This test is only aimed at samples with lower protein content than silkworm cocoons, but no experimental studies have been conducted on samples with higher protein content than silk cocoons, such as imported fish meal, blood meal, and feather meal. Therefore, I personally believe that according to the weight of the sample, the protein content and the ease of digestion, the digestion time after clarification of the digestive juice can be appropriately adjusted. For a sample with a large mass of sample, a higher protein content than silkworm cocoon, or an indigestible sample, the digestive juice can be digested for a period of time ranging from 45m in to 2h. The time can be determined by the laboratory worker according to his own work experience. And decide for yourself.
3.5 Digestion fluid transfer and constant volume
After the digestion is completed, the Kjeldahl flask is removed from the electric furnace and placed in a small iron basket to cool it. At this time must pay attention to safety, with a little thick gloves to prevent burns. After cooling to room temperature, 10 to 20 ml of distilled water was added, and the mixture was cooled to room temperature and transferred to a 100 ml volumetric flask. The retransfer is to reduce the loss error. A funnel should be added to the volume bottle mouth, and the flask and funnel should be washed several times with a small amount of distilled water (that is, a small number of times). Otherwise, the measurement result will be low due to accidental errors. Do not immediately set the volume after the transfer is complete. Cool to room temperature and then set the volume. If it is not cooled and the volume is constant, the volume will be reduced after cooling, so that the amount of sample decomposing liquid sucked will be large, and the measurement result will be high. If the digestion solution is not cooled after digestion, if it cannot be stored on the same day and it needs to be stored overnight, a small amount of distilled water should be added to the Kjeldahl flask to prevent the crystallization of the digestion solution. Then, the Kjeldahl flask mouth should be wrapped in plastic cloth to prevent absorption of air. Ammonia affects the accuracy of the results. If the crystallization of the digestive juice is not easy to transfer, the Kjeldahl flask can be heated on the electric furnace until the crystals are melted and then transferred.
4 Determination of crude protein: distillation
4.1 The volume and acidity of water in the steam generator
The volume of water in the steam generator should be controlled at about 2/3 of its volume and control the liquid level. Excessive volume of water can easily cause workers to evaporate from the glass tubes in the steam generator after boiling; too little gas pressure can easily generate or vaporize steam generators to cause accidents. A few drops of sulfuric acid and a few drops of the mixing indicator are added together with the addition of water, and the water is kept purple all the time to prevent the ammonia nitrogen in the water from overflowing and affecting the measured value.
4.2 Hermeticity of distillation apparatus
Be sure to check the airtightness of the distillation apparatus before starting the distillation operation. The interface of each catheter and the rubber tube used for a long time should be examined with emphasis. The glass beaker mouth with added digestive liquid and alkaline liquid should be tightened and sealed to prevent ammonia gas from escaping. While checking the airtightness, the condensate switch should also be opened and a certain flow rate must be maintained, otherwise the cooling is not complete. This is an easily overlooked issue, especially for beginners.
4.3 Accurate removal of sample decomposition fluid
Before removing the sample decomposing fluid, shake the volumetric flask first and then pipette the 10m L pipette with the required sample decomposing fluid for 2 to 3 times, and then remove it. The pipette should be perpendicular to the ground when removing, and the line of sight should be parallel to the scale line of the pipette. After aspirating the sample solution, the pipette is placed in the reaction chamber and naturally flows into the reaction chamber. Avoid placing the pipette into the small glass cup and pipetting the pipette with the suction earball. Finally, rinse the small glass bulb with a small amount of distilled water and plug the rod-shaped glass stopper.
4.4 The amount of sodium hydroxide solution added
In the distillation process, the solution in the reaction chamber should be kept alkaline, and the amount of alkali added is generally about 10m L. If the amount of sulfuric acid added during digestion is too much, the amount of 40% sodium hydroxide solution must increase accordingly. In addition to the reaction with sulfuric acid and copper sulfate, it is guaranteed that there is a sufficient amount of alkali to convert the ammonia radical to ammonia. The alkali added is first placed in a small glass bulb, slowly rotating the rod-shaped glass stopper, so that the alkali slowly flows into the reaction chamber, and then the small glass bulb is added with a distilled water liquid seal, and it is not allowed to lift the rod-shaped cork stopper and add alkali - especially at the beginning Scholars, this will cause the ammonia generated after the reaction to escape from the glass, resulting in low levels of the measured protein.
4.5 Distillation Speed ​​and Air Flow
The gas flow generated during distillation must be uniform, avoid heat instability, half-way interruption or lack of steam, otherwise the reaction chamber temperature will decrease, easy to produce backward suction; the gas flow is too fast for the absorption of boric acid. The speed of the distillation is related to the flow of the cooling water and the temperature of the circuit, in addition to the flow. Distillation rate is fast, and the reaction solution is easily brought out by the steam to make the measurement result high. When the distillation rate is slow, ammonia is not completely distilled and the result is low. What kind of distillation rate is appropriate? According to Jia Yanling and Leng Bozhong (2000), it has been proved that the distillation speed should generally be controlled between 5 and 7 mL per minute.
4.6 Distillation time
Distillation time is usually 5m in. After adding the sample decomposition solution and sodium hydroxide, distill 4m in. After the end of the condensate tube is separated from the boric acid surface, distill it for 1m in. However, the standard does not stipulate when to start the timing. According to the test of Xie Wenfei (2003), it is proved that there is no effect on the result after distilling 1 to 2 minutes after the distillation is completed for 4 minutes, so it can be started when the boric acid has just appeared green. Chronograph distillation for 4 min (a green indication of boric acid has begun). Finally whether the distillation is fully detectable after 4 min using pH test paper. The boric acid absorption temperature should also be taken into account during distillation. Boric acid is a very weak acid and only acts to absorb ammonia, but it should be noted that the temperature of the boric acid absorption solution should not exceed 40°C, otherwise the absorption of ammonia is weakened and the result is low.
4.7 Cooling and washing of the reaction chamber
First, after the distillation is completed, the ends of the condenser tubes are rinsed with distilled water, and then the flasks are removed for titration. When cleaning the reaction chamber, the valves on both sides of the distillation flask must not be closed at the same time to avoid explosion. The cleaning of the reaction chamber must be thorough. The first is to prevent the residual liquid from affecting the accuracy of the next measurement. The second is to cool the reaction chamber. If the temperature of the reaction chamber is very high, a large amount of heat released from the reaction of the added sodium hydroxide and sulfuric acid is prone to bumping, and the reaction solution is flushed into the condenser tube, resulting in failure of the experiment. In addition, the temperature of the reaction chamber is high and the reaction is violent. Some ammonia boric acid absorption is incomplete and escapes, and the measured result will be low. The general reaction room temperature can be reduced to not hot.
4.8 Cone-shaped bottle pretreatment
Erlenmeyer flasks are often washed with detergent and rinsed with tap water and distilled water. Washing powder is not easy to clean thoroughly, resulting in the environment of the conical flask's inner wall being more alkaline; in addition, the p H of tap water varies from place to place, and the distilled water p H produced sometimes deviates from neutrality, which will lead to biased measurement results. It is recommended that the conical flask be pretreated before use. The method is: rinse the conical flask with 20mL distilled water, 2 drops of mixing indicator, and then use 0.02mol • L -1 hydrochloric acid solution or 0.02mol • L -1 The sodium hydroxide solution was titrated until it turned from blue to grayish red, and then the liquid was poured off. Then, 25 to 35 ml of boric acid absorption solution and 2 to 3 drops of mixing indicator were added. If the boric acid is blue after the addition of the indicator, it indicates that the boric acid is mixed with alkaline substances or distilled water is alkaline, and the boric acid solution needs to be re-formulated. The pretreatment of conical flasks is relatively tedious, but it can reduce the influence of pH deviating neutrality on the measurement result due to the conical flask itself.
4.9 Titration
Titration is the last step in the entire experiment and it is very important, otherwise the entire experiment will be abandoned. After the titration, shake the hydrochloric acid standard bottle, rinse the acid burette for 2 to 3 times, add the hydrochloric acid standard solution to remove the air bubbles, record the scale value, and start the titration. Titration must be controlled when the titration rate, until the receiving liquid gradually changed from light green to colorless to slow down the titration rate, adding hydrochloric acid standard solution dropwise, the receiving solution becomes light red when the end of the titration, do not drop excessive.
5 Determination of crude protein: blank assay
5.1 Determination of Reagent Blanks
Another one Kjeldahl flask or digestive tube, add copper sulfate, anhydrous potassium sulfate (or anhydrous sodium sulfate), concentrated sulfuric acid, in which the amount of each drug added is the same as when the sample is digested, heated to digest the solution Transparent blue-green. The subsequent steps are exactly the same. This corrects errors that occur with reagent impurities.
5.2 Determination of sample blank
Weigh 0.5g of analytically pure sucrose in place of the sample, after which the amount of each reagent and the procedure used are the same. The standard is that the blank measurement consumes 0.1 mol • L -1 hydrochloric acid standard solution volume does not exceed 0.2mL; consume 0.02mol • L-1 hydrochloric acid standard solution volume does not exceed 0.3mL. The above two blank determinations do not need to be performed every time, and can be operated once every other time. However, blank assays must be performed when changing reagents and standard solutions. If the blank value exceeds the standard, it is generally caused by the reason that the used reagent level is not enough or impure, the distilled water is not pure, and the glassware is not cleaned cleanly.
6 Determination of Crude Protein: Detection of Distillation Devices and Operational Accuracy
At the same time as the sample test, 0.2000g of analytically pure ammonium sulfate was accurately weighed instead of the sample, and the sample was directly equilibrated to a volume of 100m L. After distillation and titration, the ammonium content of ammonium sulfate was measured to be 21.19% ± 0.2% to calibrate the distillation apparatus. The airtightness and alkali addition, distillation and titration steps are correct.
7 Determination of Crude Protein: Calculation of Conversion Factor During Calculation
When we calculate the protein content, we multiply the coefficient by an average factor of 6.25. If the nitrogen content in the crude protein has not been determined, it can be multiplied by the average coefficient, which is not appropriate for the determination already made. Soybean meal is taken as an example: If soybean meal is measured and soybean meal is the main factor When crude protein content of concentrated feed of raw materials is used as a protein conversion factor of 6.25, it is 0.54 times larger than the actual soybean conversion factor of 5.70, and the relative error is about 8%. Therefore, the actual coefficient is used for the already determined conversion factor. To convert, for example, buckwheat, corn coefficient 6.00, soybean, peas, beans, oats, wheat, rye, arrowweed and other factors 5.70, soybeans coefficient 5.71, barley coefficient 5.83, cottonseed, sesame, sunflower seed coefficient For 5.30, peanuts have a coefficient of 5.46, full-fat soybean meal has a coefficient of 5.72, and milk has a coefficient of 6.38. The above are some experiences and experiences in the determination of protein content in feeds by the Kjeldahl method and are discussed together with everyone.