Details of crude protein determination by Kjeldahl method

More than 90% of crude protein detection uses Kjeldahl method, which is a relatively complicated process. The following details need to be noted.

1. The national standard requires that the sample be crushed through 40 mesh for two reasons: 1. It is easy to digest; 2. The sample is more uniform and the parallel results are better.

2. Generally, the sample weight is preferably 0.3g-0.5g, and the sample weighing balance is a one-tenth of a balance, retaining four decimal places.

3. It is usually necessary to add potassium sulfate or sodium sulfate to increase the boiling point of the digestion solution (290℃→400℃), and copper sulfate is added as a catalyst. Sodium sulfate is much cheaper than potassium sulfate, and sodium sulfate is generally used instead of potassium sulfate.

Digestion process:

4. Do not set the temperature directly to 420℃. This will cause a large amount of ammonia to be generated when the temperature rises rapidly, thus causing the problem of ammonia escape. The reason is that the crude protein digestion process involves two influencing factors: the gradual decrease in sample concentration and the rapid increase in temperature causing the reaction rate to rise rapidly. However, if the temperature rises too quickly at a certain moment, it is very easy for ammonia to be generated in large quantities and not have time to combine with sulfuric acid and escape. It is recommended to increase the temperature in stages, that is, to increase the temperature from low heat (lower temperature) to high heat (higher temperature) during the digestion process.

5. Setting the digestion temperature. Generally, the temperature is set in a staged heating mode, starting from 200℃ and the highest setting is 420℃ required by the national standard (of course, depending on the sample and the aging of the digestion furnace, it is okay to increase the temperature by 10-20℃). Generally, 3 hours of digestion from the beginning of digestion to the end of high temperature insulation is enough. Of course, depending on the sample, the optimal mode of the digestion furnace heating program can be adjusted through the measurement data results.

6. For example, if you use an 8-hole digestion furnace to digest samples, but only digest 4 samples at a time, it is recommended to place 4 in the middle hole position. After all, the heat dissipation in the middle is definitely not as fast as the heat dissipation of the holes on the side, so the heating and heat preservation effect will be better.

7. When digesting protein in a digestion furnace, try not to digest it open. The manufacturer will be equipped with an exhaust hood, and the exhaust hood should be covered during digestion. If there is no exhaust hood, it can generally be solved by making the exhaust hole of the digestion tube smaller, such as placing a glass funnel on the mouth of the digestion tube. Open digestion is prone to cause less digestion liquid, the main reason is that concentrated sulfuric acid cannot be effectively refluxed and evaporated.

8. Protein digestion is divided into tap water jet pump exhaust gas and waste discharge device. Among them, tap water exhaust has stronger exhaust and suction capacity, which means that the faucet should be opened to an appropriate size according to the water pressure, so that the exhaust gas generated during digestion can be normally extracted. But at the same time, the digestion furnace should be placed in a fume hood as much as possible.

So the main problem of digestion is either incomplete digestion or ammonia escape. Therefore, digestion verification is a very important part.

Distillation process:

9. The sample needs to be preheated before distillation. The purpose of preheating is to fill each pipe with reagent liquid, and the generated steam must be sufficient and stable. The preheating steps are to take an empty digestion tube, add some distilled water to the instrument, add the required reagents, and then turn on the steam heating, once every 5 to 8 minutes, and preheat 2 to 3 times.

10. The amount of alkali solution needs to be sufficient. The amount of alkali solution can be more, but not less. After the sample turns black after adding alkali, add 5-10 ml of alkali solution. For example, some instruments add alkali solution by time. If 10S can make the sample turn black, it is better to set the alkali addition time to 12s. Otherwise, because the alkalinity is not strong, ammonia is not easy to evaporate.

11. Prevent the digestive fluid from reacting violently when adding alkali solution. You can turn on the steam heating first to make the steam outlet emit a small amount of gas, and then start to add alkali by turning on the alkali adding switch (while the heating switch is also turned on). This will reduce the violent reaction when adding alkali solution, thereby reducing the risk of ammonia escaping and causing low results.

12. The distillation volume generally ensures that the receiving bottle reaches at least 150ml of liquid. The safest approach is to distill to 200ml. This can ensure that all ammonia is distilled out and absorbed by boric acid. You can occasionally use PH test paper to test the liquid at the receiving liquid port to determine whether it is completely evaporated. If it is blue, it is not completely evaporated. Only when it is all yellow is it complete. If there is a little blue, you need to steam for more than half a minute and then check.

13. According to the national standard requirements, when the receiving liquid reaches 150ml, the receiving liquid port can be separated from the liquid surface and rinsed with distilled water. To prevent cross contamination and liquid backflow.

14. Methyl red-bromocresol green indicators should be prepared with pure ethanol, and it is best to store them in the refrigerator after preparation. Take them out when using them and return them immediately after use. In this way, the methyl red-bromocresol green indicator changes color very sensitively at the endpoint.

15. It is recommended to use hydrochloric acid as the standard titration solution. Compared with nitric acid and sulfuric acid, hydrochloric acid has the best ionization effect in water. Generally, the appropriate concentration of hydrochloric acid solution is selected according to the protein content of the sample. The commonly used ones are 0.05mol/L and 0.1mol/L.

16. The sample should be titrated in time after nitrogen determination. It is best to complete the titration within ten minutes, and it should not exceed half an hour at most.

17. Titration endpoint color judgment: The color of the sample titration endpoint should be consistent with the blank titration endpoint to ensure the accuracy of the test. The solution changes from blue-green to gray-purple as the endpoint.

18. The nitrogen determination instrument verifies the airtightness recovery rate. After the instrument has been used for a long time, ammonium sulfate can be used to verify the airtightness, and it can be dried in an oven at 105 degrees for 2 hours. The full-volume method directly weighs 0.1-0.2g and distills directly; the trace method requires 2g to be fixed to 1000ml, and 10ml is taken each time for distillation.

In short, protein detection is a complicated process, including the preparation of reagents, sampling of samples, the reliability of instruments, the carefulness of laboratory personnel, etc. Any wrong step will lead to experimental failure, so it is also a process of slowly accumulating experience to ensure that there are no problems in every link, so that the results will naturally be accurate.