Kjeldahl method
The principle of Kjeldahl method: Protein is a nitrogen-containing organic compound. The food is heated and digested with sulfuric acid and a catalyst to decompose the protein, and the decomposed ammonia is combined with sulfuric acid to produce ammonium sulfate. Then alkalized distillation to ammonia free, absorbed with boric acid and then titrated with sulfuric acid or hydrochloric acid standard solution, according to the consumption of acid multiplied by the conversion factor, which is the protein content.
1. NH4(NH4)2SO4 is formed by ammonium nitration in organic matter under the action of strong heat, catalyst and strong H2SO4.
The reaction is:
2NH2-+H2S04+2H+=(NH4)2S04
2. Acts on base in Kjeldahl apparatus to release NH3 by distillation and collect in H3BO3 solution
The reaction is:
(NH4)2SO4+2NaOH=2NH3↑+2H2O+Na2SO4
2NH3+4H3BO3=(NH4)2B4O7+5H2O
3. Titrate with a known concentration of H2SO4 (or HCI) standard solution, calculate the amount of nitrogen based on the amount of HCI consumed, then multiply by the corresponding conversion factor to obtain the protein content.
The reaction is:
(NH4)2B4O7+H2SO4+5H2O=(NH4)2SO4+4H3BO3
(NH4)2B4O7+2HCl+5H2O=2NH4Cl+4H3BO3
The basic steps are as follows:
Sampling → Digestion → Distillation → Titration → Calculation
It consists of a digester and a fully automatic dynamometer distiller.
Digestion Furnace (exponential phenotype) is controlled by a single-chip microcomputer to further perfect the well-type heating furnace. The sample is placed in the digestion tube and inserted into the well-type heating furnace to heat to obtain the best thermal effect. The catalyst is added before the digestion to accelerate further. Digestive digestion, greatly reducing digestion time. The harmful gases such as S02 that have escaped from the digestive canal are discharged into the sewer through the evacuation three-way pipe on the digestive tract (which can also be recycled using a vacuum pump and a recovery bottle), effectively suppressing the escape of harmful gases, and omitting the experiment. A fume hood is installed in the room and the sample is digested and digested in about 40 minutes to completely digest.
1. Safety tube 2. Catheter 3. Steam-water separation tube 4. Sample inlet 5. Plug 6. Condensation tube 7. Absorption bottle 8. Insulation jacket
9. Reaction tube 10. Steam generation bottle
Kjeldahl method specific operation:
1. Sample treatment: Weigh accurately 0.2-2.0g solid sample or 2-5g semi-solid sample or draw 10-20ml liquid sample (about 30-40mg equivalent nitrogen), move into dry 100ml or 500ml bottle, add 0.2 g Copper sulphate, 6g potassium sulfate and 20ml sulfuric acid. Shake a little to put a small funnel on the bottle mouth. Hold the bottle diagonally at a 45-degree angle on the asbestos mesh with small holes. Heat it over low heat until all the contents are carbonized. After the foam is completely stopped, strengthen the firepower and keep the liquid in the bottle slightly boiling until the liquid is blue-green and clear and transparent, and then continue to heat for 0.5 hours. Remove cooling, carefully add 20ml of water, let cool, move into a 100ml volumetric flask, and use a small amount of water to wash the nitrogen bottle, wash solution into the volumetric flask, add water to the mark, mix and spare. Take the same amount of copper sulfate, potassium sulfate, and ammonium sulfate in the same way as the sample to make a reagent blank test.
2. Install the nitrogen device as shown in the figure, add 2 drops of methyl red indicator and a few milliliters of sulfuric acid to the water inside the water vapor generator to keep the water acidic and add a few glass beads to prevent bumping. Controlled by a pressure regulator, the boiled water vapor is heated to generate water in the bottle.
3. Add 10 ml of 2% boric acid solution and one indicator of mixing indicator to the receiving flask, and insert the lower end of the condenser into the liquid surface. Pipette 10.0 ml of sample digestion solution from the small glass into the reaction chamber and wash it with 10 ml of water. The beaker was allowed to flow into the reaction chamber and the glass stopper of the small glass was plugged. Pour 10 ml of 40% sodium hydroxide solution into a small glass, lift the glass stopper and allow it to slowly flow into the reaction chamber. Immediately plug the glass lid and add water to the small glass to prevent air leakage. The screw clamp is clamped and distillation begins. Vapor is passed into the reaction chamber to allow the ammonia to pass through the condensing tube and into the receiving flask and is distilled for 5 minutes. Move the receiving bottle so that the lower end of the condenser exits the tank and distill for another 1 minute. Then flush the outside of the lower end of the condenser with a small amount of water. The receiving flask was removed and the endpoint was set to 0.01N sulfuric acid or a 0.01N hydrochloric acid standard solution set to gray or blue-purple.
At the same time draw 10.0ml reagent blank digestive juice according to 3 operation.
Calculation:
X = ((V1-V2)*N*0.014)/( m*(10/100)) *F*100%
X: percentage of protein in the sample, g;
V1: The volume of the standard sample of sulfuric acid or hydrochloric acid consumed, ml;
V2: Reagent blank consumption of sulfuric acid or hydrochloric acid standard solution volume, ml;
N: equivalent concentration of sulfuric acid or hydrochloric acid standard solution;
0.014: 1N sulfuric acid or hydrochloric acid standard solution 1ml is equivalent to the number of grams of nitrogen;
m: mass (volume) of the sample, g (ml);
F: Nitrogen is converted into a protein coefficient. The nitrogen content in protein is generally 15-17.6%, calculated as 16% multiplied by 6.25 which is protein, dairy products is 6.38, flour is 5.70, corn, sorghum is 6.24, peanut is 5.46, rice is 5.95, soybean and its products It was 5.71, meat and meat products was 6.25, barley, millet, oats, rye were 5.83, and sesame and sunflower were 5.30.
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