At present, there are four ancient classical methods commonly used, namely nitrogen determination, biuret method (Biuret method), Folin-phenol reagent method (Lowry method) and ultraviolet absorption method. There is also a new assay commonly used in the last decade, the Coomassie brilliant blue method (Bradford method), and the recently widely used protein quantification method, the BCA method. The Bradford method and Lowry method have higher sensitivity, 10-20 times more sensitive than UV absorption method and 100 times more sensitive than Biuret method. Although the nitrogen determination method is relatively complicated, it is more accurate and the protein determined by the nitrogen determination method is often used as a standard protein in other methods. 6 O! d- J2 r) @; g* t& J4 m! I Kjeldahl method This method was invented by Kjeldahl in 1883. At that time, Kjeldahl only used H2SO4 to decompose the sample to quantify the pro in the grain. He only knew how to use H2SO4 to decompose the sample, but could not clarify the reaction course of H2SO4 decomposing the organic nitrogen compound to produce ammonia. So only using H2SO4 to decompose the sample takes a long time, and later Gunning joins the improvement. His improvement is to add K2SO4 during digestion to increase the boiling point, which accelerates the decomposition rate because the temperature rises from the original boiling point of 380 to 400 °C. Since the speed is increased at less than 67°C, the Kjeldahl method is still in use today. When we test pro in foods, we often limit ourselves to measuring total nitrogen, and then multiplying 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 or micro nitrogen determination method. The first step is to digest: (1) Digestion: The sample is heated and digested with sulfuric acid, and sulfuric acid dehydrates the organic matter. And to destroy organic matter, so that the organic matter C, H oxidized to CO2 and H2O vapor escape, and pro is the decomposition of nitrogen, then combined with sulfuric acid into ammonium sulfate, leaving in the acidic solution. (2) Adding potassium sulphate during the digestion process can increase the temperature and accelerate the decomposition of organic matter. It reacts with sulfuric acid to form potassium bisulphate, which can increase the reaction temperature. Generally pure sulfuric acid heats up the boiling point of 330°C. After adding potassium sulphate, the temperature can reach 400°C. Accelerated 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 sulphate cannot be added too much, otherwise the temperature is too high and the resulting ammonium bisulphate will also decompose, releasing ammonia and causing losses. In order to accelerate 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) 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. (2) Absorption and titration: The ammonia evolved during the distillation can be absorbed by a certain amount of standard sulfuric acid or a standard hydrochloric acid solution, and then the excess sulfuric acid or hydrochloric acid solution can be back-titrated with a standard sodium hydroxide solution to calculate the total nitrogen amount. Semi-micro or trace nitrogen is usually absorbed in boric acid solution and then 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. Steps: Accurately weigh 0.50-2.00g of the sample → In a 500ml Kjeldahl bottle → Add 10g of anhydrous K2SO4 → Add 0.5g of CuSO4 → Add 20ml of H2SO4 → Heat in a small amount of heat in a fume hood. After the foam disappears, increase the firepower. After digestion to transparent no black particles, the bottle is shaken to dissolve the wall carbon particles in sulfuric acid → continue digestion for 30 minutes → until the sample solution is in a green state, stop digestion, cooling → add 200 ml of water → connect the distillation apparatus → use boric acid As an absorbent → Add a few beads in a K bottle and 80ml of 50% NaOH → Immediately fix a ball of nitrogen → Heat → When the residue in a K bottle is reduced to one-third, remove it with water → Use 0.1 Titrate with N HCl. N(V2-V1)0.014 Total nitrogen content = (N(V2-V1)×0.014)/W × 100 0.014----milligrams of nitrogen equivalents 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 eggs K=6.7 (N=14.8%) Soybean product K=6.0 (16.7%) K=6.25 (N=16%) K-replacement number The role of various reagents: H2SO4: A: Dehydration carbonizes organics, then carbonizes organics to carbon, carbon reduces H2SO4 to SO2, and then becomes CO2 B: Oxidation C: pro and concentrated H2SO4 produce NH3↑, CO2, SO2, H2O↑ D: Ammonium sulfate generated from NH3 and H2SO4 (1) The role of CuSO4 (catalyst) CuSO4 is a red precipitate. When C is completely digested, the reaction stops, red color disappears, and it turns blue, which means that the digestion is complete, and the blue color is CuSO4. (2) The role of K2SO4 (boiling point increase) Increasing the boiling point from 330 °C to 400 °C accelerates the reaction process. (3) Selenium powder, hydrogen peroxide, and mercury oxide are all catalysts, but copper sulfate is usually used to prevent contamination. (4) The effect of 50% NaOH Here I will 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 is required to have a minimum of 500ml, preferably 800 to 1000ml. Such a K-flask is best for shortening the ammoniation time, heating uniformity and complete ammoniation. (2) Decomposition agent A H2SO4 and K2SO4 additions The amount of H2SO4 is required for organic non-decomposition, and the amount of H2SO4 to be added varies according to 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 is recognized as There is also a ratio: K2SO4:H2SO4=10:20ml B Catalyst Used as catalysts are Hg, HgO, Se, selenium compounds, CuSO4, TiO2, toxic to Hg, HgO but results are good, Se and CuSO4 results are one, TiO2, the result is low, using different catalysts will be the digestion time Differently, HgO digests wheat for 38, Se digests wheat with CuSO4 55, and TiO2 digests wheat 70. Therefore, the type of catalyst should be specified when giving the measurement result. (3) Strength of heat source The intensity of the heat source during digestion is greatly 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. (4) Distillation and absorption of ammonia and titration There are two types of distillation: 1. Direct distillation (device is simple and accurate) 2. Steam distillation Distillation plus NaOH is 50%, the amount added is 4 times the amount of H2SO4, the amount of sulfuric acid is 12 ml, then the NaOH is 12 x 4 = 48 ml, and is generally higher than this theoretical value, ie added to 50-55 ml, if the amount of NaOH is increased Not enough to become H2S, H2S is strong acid, making the color red. Absorbent fluids are: 1 Standard H2SO4 back titration with standard alkali, formaldehyde red indicator 2 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 indicator color change range, and boric acid is the concentration of the absorption solution More than 3% can completely absorb ammonia, which is generally 4% during the insurance period. <6> Experiment Precautions a. Sample should be uniform, if the solid sample should be carefully studied in advance, the liquid sample should be mixed evenly. b. 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. c. When digesting, if it is not easy to be a transparent solution, the K flask can be allowed to cool, and then 2 to 3 ml of a 30% hydrogen peroxide catalyst is added to promote oxidation. d. During the entire digestion process, do not use strong fires and maintain gentle boiling. Focus the fire on 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. e. In the absence of sulphuric acid, excessive potassium sulphate will result in the loss of ammonia. Potassium bisulphate will form instead of ammonia, so when too much sulphuric acid is consumed or the fat content in the sample is too high, To add sulfuric acid. f. The mixed indicator is green in alkaline solution, gray in neutral solution, red in acidic solution, if there is no bromocresol green, 0.1% formaldehyde red ethanol solution can be used alone. g. The ammonia is completely distilled out. The pH test paper checks whether the distillate is alkaline. h. When a concentrated alkali is added to the decanter, brown precipitates are often absent. At this time, 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. i. Digestive agent can be digested green for 30 minutes. 2.K's method of determination of nitrogen 3. K's semi-micro determination method (2, 3 principle is the same) The method of operation is much the same. After digestion with a semi-micro method, the volume is 100 ml, and then absorbed by 25 ml of distilled liquid. N(V2-V1)0.014 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. 4.K's 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. The digestion device is a K-digested bottle made of high quality glass and Infrared device digestion furnace. Dihydrate acid colorimetry: 1. Principle: After the pro in the sample is converted into ammonium salt solution by H2SO4 digestion, it reacts with sodium salicylate and sodium hypochlorite at a certain acidity and temperature to produce a colored compound, which can be measured colorimetrically at a wavelength of 660 nm. The sample contains nitrogen and the protein content is calculated. 2. Method (1) Drawing of standard curve Take 6 25ml volumetric flasks 0 1 2 3 4 5 6 Separately add blank acid 2ml Separately add phosphate buffer 5ml Diluted to total volume to 15ml Separately add sodium sulfate 5ml 37C water bath 15 minutes Add sodium hypochlorite 2.5ml 37C water bath 15 minutes Remove the test solution for colorimetric comparison at 660 nm and draw a standard curve. (2) Sample processing: Accurately weigh 0.20 ~ 1.00g → In the K bottle → Add 15ml H2SO4 and 5g catalyst → Heated to boiling on the electric furnace → Increase Digestion by fire → Until dark green appears → Shake the bottle → After K bottle is completely digested → Cool down → Add water to 250 ml capacity bottle. (3) Sample measurement: Accurately absorb 10ml of the above digestion solution → In a 100ml volumetric flask → Constant volume → Accurately absorb 2ml → In a 25ml volumetric flask → Add 5ml phosphate buffer → the following procedure is the same as the standard curve With the reagent blank control, the optical density of the sample solution was measured and the nitrogen content was determined from the standard curve. (4) Calculation C×F Nitrogen content = ---------------------------× 100 W×1000×1000 C--- Determine the nitrogen content (Ug) of the sample solution from the standard curve F--- dilution of sample solution W--- sample weight (g) Pro%=total nitrogen %K (K can be 6.25, also available) 3. Note: A. The same day, the digestive juice is best measured on the same day, and the result is reproducible. If the sample solution is changed to the second day, the color change will change. B. When the chlorine source is added in the proper range of pH and reagent, the color development depends on the temperature, and the reaction temperature should be strictly controlled. C The results of this method are basically consistent with K's method. 4. Reagents (1) Chlorine standard solution: Ammonium sulphate 0.4719 g dried at 110°C for 2 hours → Constant volume 10 ml in the flask → 1 ml equivalent to this solution 1.0 mg nitrogen standard solution → formulated into 1 ml equivalent to 2.50 Ug nitrogen standard solution when used. (2) Blank Acid: Weigh 0.50g sucrose → Add 15ml H2SO4 and 5g of catalyst → Digest with sample → Constant volume 250ml → When using, absorb 10ml of this liquid → add water to 100ml for working solution. (3) Phosphate buffer: Dissolve 7.1g of disodium hydrogen phosphate → Add 38g of trisodium phosphate → Add 20g of potassium sodium potassium trioctylate → Add 400ml of water Dissolve→Filter→Also 35g NaOH dissolved in 100ml water→Cool to room temperature→Add slowly to the phosphate solution→Add Into the water diluted to 1000ml standby. (4) sodium salicylate solution: 25g of sodium salicylate and 0.15g of sodium nitroferrocyanide are dissolved in 200ml of water, filtered and diluted with water. Up to 500ml (5) Sodium hypochlorite solution: Take 4ml Antifumin Solution and dilute to 100ml with water. 5 Instruments (1) Spectrophotometer (2) Constant temperature water bath Three ultraviolet spectrophotometry 1. Principle: The aromatic ring group of pro and its degradation product has a certain light selective absorption in a certain wavelength region in the ultraviolet region. At 280 nm, the light absorption is in line with the pro concentration (3-8 mg/ml). Therefore, the determination of pro The absorbance of the solution was determined by referring to a standard sample previously analyzed by the K. D method, and the protein content was determined from the standard curve. 2. Reagents: (1) 0.1 mol/l citric acid aqueous solution. (2) 2N NaOH solution of 8 mol/l urea [(NH2)2CO]. Urea 2N sodium hydroxide solution (3) 95% Ethanol (4) anhydrous ether 3. Instruments (1) 751 UV spectrophotometer (2) Centrifuge 4. Operation method (1) Drawing a standard curve: Accurately weigh sample 2.00g, place in 50ml flask, add 0.1ml/l citric acid aqueous solution 30ml, stir for 30 minutes to fully dissolve, filter in glass centrifuge tube with four layers of gauze Centrifuge at a rate of 3,000 to 5,000 revolutions per second for 10 minutes. Aspirate 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mL of supernatant each time on six 10ml volumetric flasks, each containing 8mol/l urea hydrogen. The sodium oxide solution was shaken well for 2 minutes (if centrifuged again), the clear solution was taken in a cuvette, and the absorbance was measured at 280 nm. (doing the reference value) The standard curve was plotted by taking the absorbance on the micro-ordinate axis of the protein content in the sample determined in advance by the K-nitrogen determination method as the ordinate. (2) Sample determination Accurately weigh the sample 1.00g → in a 50ml beaker → Add 0.1mol/l citric acid solution 30ml → Stir for 10 minutes → Filter the four layers of gauze into a centrifuge tube → Set to volume with 8mol/L urea in NaOH solution, shake well The absorbance was measured at 280 nm and the pro content was determined from the standard curve. Calculation: Protein = C/W× 100 C---- pro content (mg) found from the standard curve W----measurement sample solution equivalent to sample amount (mg) Explanation: a. This method is applied to cakes, milk and soluble pro samples. When measuring cakes, the skin color should be removed. b. Temperature has an effect on pro hydrolysis, and the operating temperature should be controlled at 20~30°C. Quadruple Urea Method-Pinik Method 1 Principle: Biuret can combine with CuSO4 to form a reddish purple complex in alkaline conditions. The pro molecule contains a peptide chain similar to the biuret structure and also shows this reaction. This method is directly used to determine the pro content of solid samples such as wheat flour. But as a stabilizer for copper, sodium potassium tartrate is better than glycerin. The pro in wheat flour can be directly quantified while being extracted. 2 Reagents (1) Glycerin as a stabilizer: Take 10ml of 10N KOH solution, add 3.0ml of glycerol to 937ml of water, and vigorously stir while adding 4% CuSO450ml. (2) Sodium tartrate as a stabilizer, 10 ml of 10N KOH solution and 20 ml of 25% sodium potassium tartrate solution were added to 930 ml of water and vigorously stirred while adding 40 ml of 4% CuSO4 solution. The preparation of the above two solutions and reagents must be clarified and no copper hydroxide should be generated, otherwise it will be reconstituted. 3 methods: sample determination Standard curve drawing Accurately weigh 0.6g sample → use reagents (1) Accurately weigh 0.5g sample → use reagent (2) If reagents (2) (1) Accurately weigh → 0.5g → Centrifuge tube in 80ml → Add 1ml ClC4 → Mix → Add 50ml sodium potassium tartrate stabilizer → Centrifuge the lid for 10min (4000rpm) → Place for 1 hour → Suction mixture 15ml → 20ml centrifuge tube → Centrifuge to complete transparency → Take supernatant 5ml in → 10ml volumetric flask → Add water to constant volume → Determine the absorbance at 550nm, find the pro content from the standard curve. (2) Standard curve drawing According to the sample determination method, according to the pro content of the sample, take the centrifugation sample solution 0.0 2.0 4.0 8.0 10.0 ml in a 10 ml volumetric flask → add water to constant volume and determine the absorbance according to the sample. The pro-content in the sample was determined in advance using the K.sup.-N method as the abscissa, and the absorbance as the ordinate was plotted as a standard curve. PVA Nodular Brush Roller for Wafer and Semiconductor field, we can OEMs according to customer's request with high quality and fast lead time PVA Brush Roller,AMAT Machine Brush,PVA Brush,Wafer Brush,nodular PVA brush,nodular roller Welltronics Technology Limited , https://www.welltronics.cn
These methods cannot be applied to any form of protein under any conditions, because a protein solution is measured using these methods and it is possible to derive several different results. Each assay is not perfect and has its advantages and disadvantages. Consideration should be given to the choice of methods: 1) the sensitivity and accuracy required for the assay; 2) the nature of the protein; 3) the presence of interfering substances in the solution; and 4) the time taken for the determination.
W
Calculation:
W*10/100
Calculate total nitrogen%=(N(V2-V1)×0.014)/(W×10/100)×100