Kjeldahl method

Kjeldahl method (English: Kjeldahl method, Kayeddahl method, Kayazo method for short) is a commonly used method to determine the nitrogen content of organic compounds in analytical chemistry. This method was invented in 1883 by Kayedal. The Kjeldahl method is a common method for analyzing the nitrogen content of organic compounds. To determine the nitrogen content of organic matter, it is usually tried to convert it into inorganic nitrogen before it is measured.

First, principle:
Kjeldahl's nitrogen method firstly co-heats nitrogen-containing organic matter with concentrated sulfuric acid, undergoes a series of decomposition, carbonization, and redox reactions and other complex processes. Finally, organic nitrogen is converted into inorganic nitrogen ammonium sulfate. This process is called organic digestion. In order to accelerate and completely decompose the organic matter and shorten the digestion time, potassium sulfate, copper sulfate, mercury oxide, hydrogen peroxide, and other reagents are usually added during the digestion process. The addition of potassium sulfate can increase the boiling point of the digestive liquid and accelerate the decomposition of organic compounds. In addition, salts such as sodium sulfate and potassium chloride can also be added to increase the boiling point, but the effect is not as good as potassium sulfate. Copper sulfate acts as a catalyst. There are many types of catalysts available in the Kjeldahl method. In addition to copper sulfate, mercury oxide, mercury, selenium powder, and sodium molybdate are also available, but the most widely used ones are the effects, prices, and environmental pollution. It is copper sulfate. A small amount of hydrogen peroxide, potassium hypochlorite or the like is often added as an oxidizing agent to accelerate the oxidation of organic substances. After the digestion is completed, the digestion solution is transferred to the Kjeldahl apparatus reaction chamber, excess concentrated sodium hydroxide is added, NH4+ is converted to NH3, NH3 is driven into the bottle by distillation, excess boric acid solution is accepted, and boric acid is accepted by ammonia. Ammonium tetraborate was formed and then titrated with standard hydrochloric acid until the boric acid solution restored the original hydrogen ion concentration. The number of moles of standard hydrochloric acid consumed for titration is the number of moles of NH3, and the total amount of nitrogen can be calculated by calculation. During the titration, the endpoint of the titration was determined by the color change of the methyl red-methylene blue mixed indicator. The measured nitrogen content is the total nitrogen content of the sample, which includes organic nitrogen and inorganic nitrogen.
Take protein as an example, the reaction formula is as follows:

Digestion: Protein + H2SO4→(NH4)2SO4
+ SO2↑+ CO2 ↑+ H2O
Distillation: (NH4)2SO4 + 2NaOH → Na2SO4
+ 2 H2O + 2NH3 ↑

2NH3 + 4H3BO3 → (NH4)2B4O7
+ 5H2O

Titration: (NH4)2B4O7
+ 2HCl + 5H2O→2NH4Cl + 4 H3BO3

Proteins are a complex class of nitrogen compounds, each of which has a constant nitrogen content [about 14% to 18%, with an average of 16% (mass fraction)]. The nitrogen content measured by the Kjeldahl method is multiplied by a coefficient of 6.25, which is the protein content.

This method is applicable to the determination of samples with 0.2-1.0 mg of nitrogen.
Through this experiment, students can understand the principle of the Kjeldahl method and master the Kjeldahl method of operation.

Second, the methods and steps The organic compounds and sulfuric acid co-heat so that the nitrogen into ammonium sulfate. In this step, potassium sulfate is often added to the mixture to increase the boiling point of the intermediate product (from 169°C to 189°C). The end point of the decomposition of the sample is well judged because the mixture becomes colorless and transparent (dark at the beginning).

A small amount of sodium hydroxide was added to the resulting solution and then distilled. This step will convert the ammonium salt to ammonia. The total amount of ammonia (which is directly determined by the nitrogen content of the sample) will be determined by back titration: the end of the condenser will be immersed in a boric acid solution. Ammonia reacts with acid, while excess acid is titrated with sodium carbonate under the indication of methyl orange. The result of the titration is multiplied by a specific conversion factor to get the result.

The amount of nitrogen measured by the Kjeldahl method is generally referred to as the total Kay nitrogen amount. The total Kay nitrogen is not present. The equivalent page for English Wikipedia is Total Kjeldahl Nitrogen. . The total amount of kaupine sometimes does not really reflect the amount of protein in the sample because the portion of nitrogen measured may not be converted by the protein.

(a) Digestion
1. Prepare 6 Kjeldahl flasks and labels. Add 1.0mL of the appropriate concentration of protein solution to flasks No.1 and No.2 and No.3 respectively. The sample should be added to the bottom of the flask, and do not stick to the bottle neck or bottleneck. In turn, potassium sulfate-copper sulfate contact agent 0.3 g, concentrated sulfuric acid 2.0 mL, and 30% hydrogen peroxide 1.0 mL were successively added. Flasks Nos. 4, 5, and 6 were used as blank controls to determine trace amounts of nitrogen-containing substances that may be contained in the reagents and to calibrate the samples. In flasks Nos. 4, 5 and 6, 1.0 mL of distilled water was added instead of the sample solution, and the remaining reagents were the same as flasks Nos. 1, 2 and 3.
2. Place each flask with reagents on the digestion rack and connect the suction device. First boil and heat, the material in the flask will become carbonized and black, and produce a lot of foam. Care must be taken to prevent bubbles from escaping the nozzle. After the foam disappears and ceases to be generated, increase the firepower to keep the liquid in the bottle slightly boiling. After the solution is clarified, continue heating to slightly digest the liquid for 15 minutes. During the digestion process, the flask must be turned at any time 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 irritant. Therefore, the pump should be opened from the beginning to the end to pump tap water into the tap water. The entire digestion process should be performed in a fume hood. After digestion was complete, the flame was closed and the flask was allowed to cool to room temperature.

(b) Distillation and Absorption Distillation and absorption are performed in a micro Kjeldahl apparatus. There are many types of Kjeldahl nitrogen distillation devices, which are generally composed of three parts: steam generation, ammonia distillation, and ammonia absorption.
1. Before the instrument is installed, all parts must be cleaned by general methods. The rubber tube and plug used must be immersed in 10% NaOH solution, boiled for about 10 minutes, washed, boiled for 10 minutes, washed several times, and then installed. Fixed on a metal stand.
Before the instrument is used, all traces of the pipeline must be steam-washed to remove possible residual ammonia in the pipeline. The instrument being used can be steam-washed for 5 minutes before each sample is measured. For instruments that have not been used for a long time, repeat the steam washing, not less than three times, and check whether the instrument is normal. Carefully inspect the connections to ensure they are airtight.

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 being distilled out and affect the results, 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 cup to allow the water to flow into the reaction chamber through the tube, but do not let the water in the glass cup shine. Plug the rod-shaped glass plug to 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 quickly. Steam is evaporated from the upper port of the reaction chamber through the nitrogen ball into the cooling pipe to cool the cooling pipe. Place an Erlenmeyer bottle at the bottom to receive condensate. Measured from the start of the constant nitrogen ball, cook continuously for 5 minutes, then remove the gas lamp. After the flushing is completed, the rubber tube connecting the steam generator and the collector is clamped. Since the gas cooling pressure is reduced, the waste liquid in the reaction chamber is automatically drawn into the reaction chamber housing, and the waste liquid discharge opening clip is opened to discharge the waste liquid. Wash 2 or 3 times in this way, and then put a conical flask containing a boric acid-indicator mixture under the condenser tube so that the lower part of the condenser tube is completely immersed in the solution. Distill for 1 to 2 minutes. Observe the solution in the conical flask. Is it discolored? If it does not change color, it means that the inside of the distillation apparatus has been cleaned. Remove the Erlenmeyer flask and distill for 1~2min. Rinse the lower condenser mouth with distilled water and turn off the gas lamp. The instrument can be used for testing samples.

2. Distillation and Absorption of Inorganic Nitrogen Standard Samples

Due to the tedious operation of nitrogen sizing, in order to become familiar with the distillation and titration operation techniques, beginners should first use the inorganic nitrogen standard sample for repeated practice, and then determine the unknown organic nitrogen sample. Ammonium sulfate is usually tested three times with known concentrations.

Take five clean 100mL Erlenmeyer flasks, followed by 2% boric acid solution 20mL, methylene blue - methyl red mixed indicator (purple-red) 3 to 4 drops, cover the bottle ready for use. Take one of the Erlenmeyer flasks to receive the lower end of the condenser tube and immerse the outlet of the condenser tube 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 draw 2mL of 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 bulb 3 times with a small amount of distilled water and put it into a distillation flask. Then add 10 mL of 30% NaOH solution to the small glass bulb in the measuring cylinder and let the alkali solution slowly flow into the distillation flask. When the alkali solution has not completely flowed in, place the rod-like glass stopper tightly. About 5 mL of distilled water is added to the small glass bulb, and the glass stopper is slowly opened, so that half of the water flows into the distillation flask and half of the water remains in the small glass bulb as a water seal. The collector piston is closed and the steam generator is heated and distilled. The boric acid-indicator mixture in the Erlenmeyer flask changes from magenta to green due to the absorption of ammonia. From the time of discoloration, distill 3~5min, move the conical flask so that the liquid level in the bottle is about 1cm away from the lower end of the condensing tube, and rinse the lower end of the condensing tube with a small amount of distilled water. Continue distilling for another 1min. Remove the Erlenmeyer flask and cover it. Prepare for titration.

After the distillation is completed, remove the gas lamp, clamp the rubber tube between the steam generator and the collector, and remove the waste liquid after reaction. Rinse the small glass cup several times with water and remove the waste solution. After repeated rinsing, the next sample can be distilled. Repeat with standard ammonium sulfate twice as above. Another 2 mL of distilled water was used instead of standard ammonium sulfate for blank determination twice. The distilled flasks were titrated together.

3. Distillation absorption of unknown samples and blanks

Three samples of digested protein were used and three blank control solutions were distilled and absorbed.

Add 5mL 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 with hot distilled water 3 times, each time about 3mL of water, wash solution is poured Reaction room. 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 sticky and difficult to pour out of the Kjeldahl flask. It must be diluted with hot distilled water (5 mL). If crystals are precipitated, it must be dissolved with a slight heat, and the glass beaker should be added when hot. Into the reaction chamber. In addition, it should also be noted that if the instrument is not completely cooled before washing, the sample or the blank control solution is added immediately, otherwise the digestive solution will easily crystallize through the cooled pipe, causing blockage.
(iii) After titration samples and blanks are distilled, titrate them together.
The receiving cap was opened, and titration was performed with an acid microtiter to a standard hydrochloric acid solution of 0.0100 mol/L. When the solution is dripped into the bottle and the solution is dark gray, rinse the periphery of the Erlenmeyer flask with distilled water once. If it is green after shaking, it should be carefully dropped half a standard hydrochloric acid solution, shaking observation of the color change in the solution bottle, dark gray unchanged within a minute or two, when it is regarded as reaching the end of the titration. If pink, indicating that the titration end point has been exceeded, 0.02 mL can be subtracted from the amount of standard hydrochloric acid solution that has been titrated. The color of the final nitrogen determination endpoint of each sample must be exactly the same. The color of the solution in the receiving flask of the blank control solution was unchanged or slightly changed and no green color could be detected. Record the milliliters of standard hydrochloric acid solution for each titration for calculation purposes.

Third, results and calculations

The following formula is used to calculate the total nitrogen content of each inorganic nitrogen standard sample and unknown sample.

In the formula
WN - milligrams of nitrogen per milliliter of sample;

A - titration sample consumption of hydrochloric acid (mL);

B - titration blank consumption of hydrochloric acid (mL);

C - Determination of the amount of sample taken (mL);

0.0100 - the concentration of standard hydrochloric acid (mol/L);

14.008 - Mass per mole of nitrogen atom (g/mol).

The relative error of nitrogen content measured in three samples should be less than ±2%.

Sample Crude Protein Content = Total Nitrogen x 6.25
6.25 is the coefficient of conversion of nitrogen content to protein content. The coefficient is derived from the average protein nitrogen content of 16%. In fact, due to the different amino acid composition of various proteins, the nitrogen content is not exactly the same.

Fourth, application

The universal applicability, accuracy and repeatability of the Kjeldahl method have been widely recognized internationally. It has been determined as the standard method for detecting protein content in foods.

Tie Plate

Tie Plate,Hook Twin Tie Plate,Railway Base Plate,Railway Steel Tie Plates

Anyang Railway Equipment CO.,Ltd , https://www.ayrailwayat.com