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Kjeldahl is an instrument that calculates the protein content by measuring the nitrogen content of the sample based on the principle that the nitrogen content of the protein is constant. Because the method for calculating the protein content is called Kjeldahl method, it is called Kjeldahl instrument, also known as nitrogen analyzer, protein analyzer, and crude protein analyzer. This method was invented by Kejdal in 1883.
principle
The organic compound is co-heated with sulfuric acid to convert the nitrogen therein to ammonium sulfate. In this step, potassium sulfate is often added to the mixture to increase the boiling point of the intermediate product. The end of the sample analysis process is well judged because the mixture will become colorless and transparent (very dark at the beginning)
A small amount of sodium hydroxide was added to the obtained solution, followed by distillation. This step converts the ammonium salt to ammonia. The total ammonia amount (determined directly by the nitrogen content of the sample) is determined by the back titration method: the end of the condenser tube is immersed in the boric acid solution. Ammonia reacts with the acid, while excess acid uses sodium carbonate under the direction of methyl orange. The results obtained by titration are multiplied by a specific conversion factor to obtain the result.
Scope of application
Kjeldahl instrument uses Kjeldahl method to detect ammonia, protein nitrogen, phenol, volatile fatty acids, cyanide, sulfur dioxide, ethanol, etc. in grains, food, feed, water, soil, sludge, sediments and chemicals. . It has a very good cost performance, and only the titration process requires manual operation, which is very suitable for routine testing in laboratories and inspection agencies. Widely used in the analysis of nitrogen or protein content of food, crops, seeds, soil, fertilizer and other samples.
Steps for usage
digestion
1. Prepare 6 Kjeldahl flasks, numbered. Add 1.0 mL of the appropriate concentration of protein solution to the flasks 1, 2, and 3, and add the sample to the bottom of the flask. Do not stick it on the bottle neck or bottle neck. Then, 0.3 g of potassium sulfate-copper sulfate contact agent, 2.0 mL of concentrated sulfuric acid, and 1.0 mL of 30% hydrogen peroxide were sequentially added. The 4, 5, and 6 flasks were used as blank controls to determine the trace amount of nitrogenous substances that may be contained in the reagents, and the samples were calibrated. To the flasks 4, 5, and 6 was added 1.0 mL of distilled water instead of the sample solution, and the remaining reagents were the same as those of the No. 1, 2, and 3 flasks. 2. Place the flasks with the reagents on the digesting rack and connect the pumping device. First, heat it with a slight heat. At this time, the material in the flask becomes carbonized and black, and a large amount of foam is generated. Care must be taken to prevent bubbles from rushing out of the nozzle. After the foam disappears and stops, increase the firepower and keep the liquid in the bottle slightly boiling. After the solution is clarified, continue heating to make the digestive juice boil for 15 minutes. The flask should be rotated at any time during the digestion process so that the adhesive material on the inner wall can flow into the bottom to ensure complete digestion of the sample. The gas released during digestion contains SO2, which is highly irritating. Therefore, the pump should be turned on and the gas is pumped into the tap water from the beginning. The entire digestion process should be carried out in a fume hood. After the digestion was complete, the flame was turned off and the flask was allowed to cool to room temperature.
OMNILABD5000 series Kjeldahl nitrogen characteristics
1. The operation is extremely simple and can be quickly programmed with one-button operation. There are different language menus to choose from.
2. Equipped with optical and acoustic fault alarm system. The digestive tract is not in place or placed incorrectly, the safety door is not closed, the instrument does not start, the acid, alkali, and diluent are missing. The host can quickly detect and suspend the experiment, and the automatic alarm prompts, extremely convenient, safe and reliable.
3. Door contact safety switch.
4. Programmable reaction and distillation time.
5. Automatic steam power generation. Steam power generation can be adjusted from 40% to 100%.
6. Measurement range: 0.1-200mg
7. Nitrogen recovery rate of 99.5%
8. Repeatability ± 1%
9. Distill between standby operations.
10. Independent cleaning program.
11. Automatic and manual addition of sodium hydroxide.
12. RS232 interface.
13. Condensate consumption: 5 l / min
ZDDN-II Kjeldahl nitrogen analyzer features
1.ZDDN-II Kjeldahl nitrogen analyzer, using microcomputer for process control, including manual mode and automatic mode, can be set and switched according to your needs:
In the automatic mode: the addition of alkali, boric acid, distillation, ammonia absorption process, the volume of boric acid and alkali plus the time of distillation and absorption can be set by themselves.
In the manual mode: the three processes of adding boron, adding alkali and distilling can be manually operated, and the volume and time are controlled by themselves to meet the needs of professional users.
2. Large-screen dot-matrix liquid crystal display, full Chinese menu, touch button, easy to operate.
3. Automatic distillation control, automatic watering, automatic water level control, automatic water stop and water pressure low alarm.
4. Various safety protection: digestive tube safety door device, steam generator water shortage alarm.
5. The operating program can be stored.
6. The outer shell of the instrument is made of special spray-coated steel plate, and the working area is made of ABS anti-corrosion board and stainless steel bottom plate.
7. Anti-chemical corrosion and mechanical damage to the surface, acid and alkali resistance.
8. Water level detection, low water level alarm, automatic power off.
9. The standard does not contain the digestion furnace, the digestion furnace is optional, it is recommended to choose the C-type digestion furnace.
Distillation absorption
Distillation and absorption were carried out in a micro-Kjeldahl instrument. There are many types of Kjeldahl distillation plants, which are generally composed of three parts: steam generation, ammonia distillation and ammonia absorption.
1. Washing of the instrument
Before the instrument is installed, all parts need to be cleaned by the general method. The rubber tube and plug should be immersed in 10% NaOH solution, boiled for about 10 minutes, washed with water, boiled for 10 minutes, washed several times, then installed and fixed on an iron. On the stand. Before the instrument is used, all the traces of the pipeline must be washed with steam to remove the ammonia that may remain in the pipeline. The instrument being used can be washed for 5 minutes before each sample. For instruments that have not been used for a long time, repeat the steam wash, no less than three times, and check that the instrument is normal. Carefully inspect the connections to ensure that there are no air leaks. First, add about 2/3 volume of distilled water to the steam generator, add a few drops of sulfuric acid to keep it acidic, to avoid the ammonia in the water being distilled out to affect the result, and put a little zeolite (or capillary, etc.) to prevent explosion. Add about 20 mL of distilled water along the wall of the small glass to allow water to flow into the reaction chamber through the cannula, but the water in the glass should not be exposed, plug the rod-shaped glass stopper, keep the water seal and prevent air leakage. Immediately after the steam occurs, the switch on the waste liquid discharge pipe is closed, so that the steam can only enter the reaction chamber, causing the water in the reaction chamber to boil rapidly. The steam is distilled out from the upper port of the reaction chamber through the nitrogen-fixing ball into the condenser to be cooled, in the condensation tube. Place a conical flask on the lower end to receive the condensate. Start from the time when the nitrogen ball is hot, continue to cook for 5 minutes, then remove the gas lamp. After the flushing is completed, the connecting rubber tube between the steam generator and the collector is clamped. Since the gas cooling pressure is lowered, the waste liquid in the reaction chamber is automatically drawn into the reaction chamber casing, and the waste liquid discharge port clamp is opened to discharge the waste liquid. So clean 2~3 times, then exchange a conical flask containing boric acid-indicator mixture under the condenser tube to completely immerse the bottom of the condenser tube in the solution, distill for 1~2min, observe the solution in the conical flask. Whether it is discolored. If it does not change color, it means that the inside of the distillation unit has been cleaned. Remove the conical flask, distill for another 1-2 min, rinse the condenser bottom with distilled water, turn off the gas lamp, and the instrument can be used for sample measurement.
2. Distillation absorption of inorganic nitrogen standard samples
Due to the cumbersome operation of nitrogen determination, in order to be familiar with the operation techniques of distillation and titration, beginners should first perform repeated exercises with inorganic nitrogen standard samples, and then determine the unknown samples of organic nitrogen. It is tested three times with standard ammonium sulfate of known concentration. Take 5 clean 100mL conical flasks, add 20mL of 2% boric acid solution, 3~4 drops of methylene blue-methyl red mixed indicator (purple red), and cover the bottle for use. Take one of the conical flasks to the lower end of the condenser and immerse the outlet of the condenser in the solution. Note: The collector piston must be opened before this operation to prevent the liquid in the conical flask from sucking back. Accurately absorb 2mL ammonium sulfate standard solution into the glass cup, carefully lift the rod-shaped glass stopper to slowly flow the ammonium sulfate solution into the distillation flask, rinse the small glass cup with a small amount of distilled water 3 times, and put it into the distillation bottle. Then, 10 mL of 30% NaOH solution was added to the small glass via the measuring cylinder, and the lye was slowly flowed into the distillation bottle, and the rod-shaped glass stopper was tightly closed when the alkali solution had not completely flowed in. Add about 5 mL of distilled water to the small glass cup, and then slowly open the glass stopper, so that half of the water flows into the distillation bottle, and half is left in the small glass cup for water sealing. The collector piston is closed, the steam generator is heated, and distillation is performed. The boric acid-indicator mixture in the conical flask changes from magenta to green due to absorption of ammonia. From the time of discoloration, distill for another 3 to 5 minutes, move the conical flask so that the liquid level in the bottle leaves the lower mouth of the condenser tube about 1 cm, and rinse the lower end of the condenser tube with a small amount of distilled water, then continue to distill for 1 min, remove the conical flask, and cover it. , ready to titrate. After one distillation is completed, the gas lamp is removed, the rubber tube between the steam generator and the collector is clamped, the reacted waste liquid is removed, the small glass cup is washed several times with water, and the waste liquid is removed. After repeated rinsing, the next sample can be distilled. Repeat twice with standard ammonium sulfate as above. Another 2 mL of distilled water was used instead of the standard ammonium sulfate for the blank measurement twice. Each of the distilled conical flasks was titrated together.
3. Unknown sample and blank distillation absorption
Three samples of the digested protein were prepared, and three blank control solutions were sequentially subjected to distillation absorption. Add 5mL of hot distilled water to the digested sample or blank control solution, add it to the reaction chamber through a small glass cup, and then wash the small glass cup 3 times with hot distilled water. The water consumption is about 3mL each time, and the washing liquid is poured together. In the reaction chamber. The rest of the operation was carried out by distillation of standard ammonium sulfate. Due to the high concentration of potassium sulfate in the digestive juice, it is viscous and difficult to pour out from the Kjeldahl flask. It must be diluted with 5 mL of hot distilled water. If crystals are precipitated, it must be slightly heated and added to the glass to make it hot. Flow into the reaction chamber. In addition, it should be noted that the sample or blank control solution is added immediately when the instrument wash has not been completely cooled, otherwise the digestive juice is easily precipitated and crystallized through the cooled pipe, causing clogging.
defect
It can be known from the Kjeldahl nitrogen determination method that the Kjeldahl method is to convert the nitrogen-containing organic matter into inorganic nitrogen ammonium sulfate for detection, and obtain the measured value of the nitrogen content multiplied by a certain coefficient to obtain the protein content. Nitrogen-containing organic matter is not only protein, but also melamine. The current national standard for the addition of protein in foods and the internationally accepted method of measurement are the classical Kjeldahl method, which provides an opportunity for counterfeiters. The nitrogen content of the protein is not more than 30%. The biggest characteristic of melamine is the high nitrogen content (66%). It is colorless and odorless after being dissolved in water, that is to say, adding melamine in a cup of clear water, and then using Kjeldahl The nitrogen method was tested and the results showed that it contained protein. Since the "Kjeldahl method" can only measure the nitrogen content, and can not identify the chemical substances in the feed, the melamine-added milk powder can theoretically measure a higher protein content.
application
The universal applicability, accuracy and repeatability of the Kjeldahl method have been widely recognized internationally. It has been identified as the standard method for detecting protein content in foods. However, this method does not give a true protein content because the nitrogen measured may not only be converted from protein. This can be reflected in food safety incidents such as the 2007 US pet food contamination incident and the 2008 China tainted milk powder incident: melamine, a substance with a high nitrogen content, was added to food to counterfeit high nitrogen content. . In addition, this method requires many different correction factors due to different amino acid sequences. Moreover, the Kjeldahl method also requires the use of concentrated sulfuric acid and a longer period of heating (generally, greater than one hour). This also makes the Dumas method sometimes more convenient when roughly measuring protein content.
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