There are many kinds of foods, and the pro is different in the nature and content of foods, and other ingredients, such as carbohydrates, fats and vitamins, interfere with many components. Therefore, pro is usually digested into ammonium salt using the classical Kjeldahl method. Distillation, absorption with a standard acid solution, determination with a standard acid or base, and the content of pro from the nitrogen content of the sample. Because the pro content in food is divided into Kjeldahl constant method, semi-micro method and trace method, their basic principles are the same. When we test pro in foods, we often limit ourselves to measuring total nitrogen, and then multiplied by pro to calculate the protein content, which actually includes non-proteins such as nucleic acids, alkaloids, nitrogen-containing lipids, phytochromes, and nitrogen-containing pigments. Nitrogen compounds are called crude pro.
1 Kjeldahl constant nitrogen method
Their principle is the same regardless of the constant, semi-micro and micro nitrogen determination method. The first step is digestion: the sample is heated and digested with sulfuric acid, and sulfuric acid dehydrates the organic matter. And the destruction of organic matter, the organic matter in the oxidation of C, H to CO2 and H2O vapor, and pro decomposition of nitrogen, then combined with sulfuric acid into ammonium sulfate, leaving in the acidic solution. Adding potassium sulfate during the digestion process can increase the temperature and accelerate the decomposition of organic compounds. It reacts with sulfuric acid to form potassium bisulfate, which can increase the reaction temperature. Generally pure sulfuric acid heats up the boiling point of 330 °C. After adding potassium sulfate, the temperature can reach 400 °C and accelerates. The entire reaction process. In addition, sodium sulfate can also be added, and potassium salts can be hydrogenated to increase the boiling point. The reason is that sulfuric acid is continuously decomposed during the digestion process, and the concentration of potassium sulfate is increased and the boiling point is increased by the escape of moisture. Accelerated organic decomposition. However, the amount of potassium sulfate added can not be too large, or the temperature is too high, the resulting ammonium bisulfate can also be decomposed, releasing ammonia and causing losses.
In order to speed up the reaction process, copper sulfate, mercury oxide or selenium powder is also added as a catalyst and a small amount of hydrogen peroxide is added. Potassium hypochlorite is used as an oxidant. However, copper sulfate is usually used to prevent contamination. Therefore, after the organic matter is completely digested, copper sulfate appears blue-green, which has a catalytic function and can also serve as an alkaline reaction indicator.
1.1 Distillation
Ammonium sulfate in the sample solution releases ammonia under alkaline conditions. In this operation, the sodium hydroxide solution is added excessively, and the ammonia in the sample solution is prevented from escaping. Absorption and Titration: The ammonia evolved during the distillation can be absorbed by a certain amount of standard sulfuric acid or standard hydrochloric acid solution, and then the excess sulfuric acid or hydrochloric acid solution can be back-titrated with standard sodium hydroxide solution to calculate the total nitrogen. Semi-micro or trace nitrogen is usually absorbed by boric acid solution, and then directly titrated with standard hydrochloric acid. Boric acid is slightly acidic. Titration with acid does not affect the color change reaction of the indicator. It absorbs ammonia.
1.2 Operation steps
Accurately weigh the sample (0.50 to 2.00) g → in a 500 ml Kjeldahl bottle → add 10 g of anhydrous K2SO4 → add 0.5 g of CuSO4 → add 20 ml of H2SO4 → heat in a small amount of heat in a fume hood until the foam disappears and increase the firepower. After digesting to transparent no black particles, the bottle is shaken so that the bottle wall carbon particles are dissolved in sulfuric acid → continue to digest for 30 minutes → until the sample solution is green, stop digestion, cooling → add 200ml of water → connect the distillation device → use Boric acid is used as an absorbent → add beads and 80ml of 50% NaOH in the K bottle → immediately fix the nitrogen ball → heat → until the residual solution in the K bottle is reduced to 1/3, and then remove and rinse with water → use 0.1N HCl titration.
Calculation: Total Nitrogen % = (N (V2 - V1) × 0.014) / W × 100
0.014 --- milligrams of nitrogen equivalent
Pro % = Total Nitrogen % × K
Dairy products K = 6.38 (N = 15.7%)
Wheat flour K = 5.79 (N = 17.6%)
Animal glue K = 5.6 (N = 18.0 %)
Ice Egg K = 6.7 (N = 14.8%)
Soybean product K = 6.0 ( 16.7%) K = 6.25 (N = 16%)
K - change number
1.3 The role of various reagents
Concentrated H2SO4: (1) Dehydration carbonizes organics, then carbonizes the organics to carbon, and carbon reduces H2SO4 to SO2, which itself becomes CO2. (2) Oxidation. (3) pro and concentrated H2SO4 produce NH3 ↑, CO2, SO2, H2O ↑. (4) NH3 and H2SO4 produce ammonium sulfate.
CuSO4: Catalyst, CuSO4 is a red precipitate. When C is completely digested, the reaction is stopped, red color disappears, and it turns blue, that is, the digestion is complete and the blue color is CuSO4. K2SO4: Boiling point, boiling point increased from 330 °C to 400 °C to accelerate the reaction process.
Here are some aspects to explain the factors that affect the completeness and speed of ammoniation:
(1) K-flask and sample volume
If a sample of 1g or more is called, a K-flask needs a minimum of 500ml, and (800-1000) ml is better. Such a K-flask is best for shortening the ammoniating time, heating uniformity and complete ammoniation.
(2) Decomposition agent
The amount of H2SO4 and K2SO4 added. The amount of H2SO4 is required for organic non-decomposition, and the amount of H2SO4 added depends on the type of organic material. If the sample contains high amounts of lipids, more H2SO4 is added. In order to increase the decomposition temperature, a large amount of K2SO4 is added, but not too much. Too little, too little ammoniation is insufficient. The addition ratio of K2SO4 and H2SO4 is: 1g sample K2SO4: H2SO4 = 7g: 12ml This proportion is used at home and abroad, and it is recognized as a ratio: K2SO4: H2SO4 = 10: 20ml
(3) Catalyst
Used as catalysts are Hg, HgO, Se, selenium compounds, CuSO4, TiO2, toxic to Hg, HgO but good results, Se and CuSO4 results are one, TiO2, the result is low, using different catalysts will be digestion time In contrast, HgO digests wheat for 38, Se digests wheat with CuSO4 55, and TiO2 digests wheat 70, so the type of catalyst should be specified when giving the results.
(4) Intensity of heat source
The intensity of heat source during digestion is related to rapid digestion and complete ammoniation. Even if K2SO4 is added in a large amount, if the heat source is weak, it is meaningless. If the heat source is too strong, H2SO4 is lost and the ammonia recovery rate is low. The size of the neck, the thickness of the neck, and the length of the neck are also related to the strength of the heat source.
(5) Distillation and absorption of ammonia and titration
There are two types of distillation: direct distillation (convenient installation, good accuracy) and steam distillation. Distillation plus NaOH is 50%, the amount added is 4 times the amount of H2SO4, the amount of sulfuric acid is 12ml, then the NaOH is 12 × 4 = 48ml, and is generally higher than this theoretical value, ie added to (50-55) ml, if When the amount of NaOH is not enough, it becomes H2S, and H2S is a strong acid that turns the color red. Absorbent solution: standard H2SO4, back titration with standard base, formaldehyde red indicator; boric acid, titration with HCl, mixed indicator. Boric acid is currently used to absorb liquid, with boric acid instead of H2SO4, which can be omitted in the back titration, H2SO4 is a strong acid, more stringent requirements, and boric acid is a weak acid, in the titration, does not affect the range of indicator color, and boric acid is the concentration of the absorption solution More than 3% can completely absorb ammonia, which is generally used for the insurance period 4%.
1.4 Experimental Considerations
(1) The sample should be uniform. If the solid sample should be finely studied in advance, the liquid sample should be mixed evenly.
(2) When the sample is placed in the K-flask, do not adhere to the bottleneck. In case the adhesion can be slowly washed down with a small amount of water to avoid incomplete digestion of the sample, the result is low.
(3) If it is not easy to present a clear solution during digestion, cool the K flask and add 30% hydrogen peroxide (2 to 3) ml to promote oxidation.
(4) During the entire digestion process, do not use strong fires and maintain gentle boiling so that the firepower is concentrated at the bottom of the K-flask so that the proteins attached to the walls do not cause loss of nitrogen in the absence of sulfuric acid.
(5) In the absence of sulphuric acid, excessive potassium sulphate will cause loss of ammonia, which will form potassium bisulphate instead of ammonia, so when excessive sulphuric acid substrate is consumed or fat content in the sample is too high To add sulfuric acid.
(6) The mixed indicator is green in alkaline solution, gray in neutral solution, red in acid solution, if there is no bromocresol green, 011% formaldehyde red ethanol solution can be used alone.
(7) Whether the ammonia is completely distilled or not, pH test paper checks whether the distillate is alkaline.
(8) When a concentrated alkali is added to a decanter, brown precipitates are often absent. In this case, the decomposition accelerator reacts with the added copper sulfate to form copper hydroxide, and after heating, it decomposes to form a precipitate of copper oxide. In some cases, Cu ions and ammonia react to form a dark blue complex.
(9) After the digestive agent is green, continue digesting for 30 minutes.
2 Kjeldamin and semi-micro determination method
The principle of the two is the same, and the operation method is similar. After the semi-micro method is digested, the volume is set to 100 ml, and then absorbed by 25 ml of distilled absorption liquid. Calculate the total nitrogen % = (N (V 2 - V 1) × 01014) / (W × 10/100) × 100
For the micro-determinator method, the instrument has been improved, the sample solution has less sample, and there is less distillate in the distillation. The others are basically the same.
3 K automatic nitrogen determination
The principle is the same as above. The instrument uses K's automatic nitrogen determination device: The device has an automatic alkali distillation device, an automatic absorption and titration device, and an automatic digital display device. Digestion device: K-digested bottle made of high quality glass and Infrared device digestion furnace.