PH buffer solution preparation

The main components of the buffer solution are hydrochloric acid, sodium acetate, acetic acid, ammonium acetate, ammonium chloride, ammonia, etc. To remove carbon dioxide, carbon dioxide can react with acetate ions to generate acetic acid. In deionization, metal ions can react with hydrochloric acid to form salts and change the pH value of the buffer.

Not only do you need to use deionized water (purified water) to prepare standard buffers, but solutions prepared in the laboratory generally use at least deionized water, and some have higher requirements. The removal of carbon dioxide in the water is to prevent the acidity of carbon dioxide from affecting the pH value of the standard buffer solution (although the addition of a small amount of acid and alkali has little effect on the pH value of the buffer solution, the buffer solution used for calibration requires relatively high accuracy. Various ions in ordinary water and a small amount of carbon dioxide will affect the accuracy of the pH value of the buffer solution, and it is necessary to pay attention to the impact of temperature on the pH value and cannot be ignored) This post was last edited by flks224086 on 2009-4-7 20:55 ]

Buffer solutions used to calibrate pH meters must be free of CO2. Other commonly used buffer solutions such as ammonia buffer solution and acetate buffer solution are not necessary.

Deionized water or distilled water should be acceptable to ensure the "purity" of the solution. Removing carbon dioxide eliminates the impact on the buffer solution. Carbon dioxide dissolves in water and is acidic, which will affect the pH value of the buffer solution!

After your water is heated and then cooled, carbon dioxide will be dissolved in it, so you must be careful to pour the water into a container with a soda-lime buffer tube while it is hot, otherwise it will be a wasted effort. Wait for the water to cool down before preparing it quickly, and then put it in a container with a soda-lime buffer tube. However, generally speaking, the pH requirements are not so strict, it is just about right.

Deionized water has no impurities. Carbon dioxide affects the pH value. What I know about the preparation of commonly used pH buffer solutions and pH value. 1.7 2 Glycine-hydrochloric acid dissolve 150 g of glycine in 500 ml of water, add 480 ml of concentrated hydrochloric acid, and add water to dilute to 1L 2.3 3 Monochloroacetic acid-sodium hydroxide dissolve 2 g of monochloroacetic acid in 200 ml of water, add 40 g of NaOH, and after complete dissolution, add water to dilute to 1 L 2.8 4 Potassium hydrogen phthalate-hydrochloric acid 25.0 ml 0.2 mol/L potassium hydrogen phthalate solution and 6.0 ml 0.1 mol/L HCl. Mix evenly and add water to dilute to 100 ml 3.6 5. Potassium hydrogen phthalate-sodium hydroxide. Mix 25.0 ml 0.2 mol/L potassium hydrogen phthalate solution and 17.5 ml 0.1 mol/L NaOH. Add water to dilute to 100 ml 4.8 6 Hexamethylenetetramine - Hydrochloric acid Dissolve 40 g of hexamethylenetetramine in 200 ml of water, add 10 ml of concentrated HCl, and add water to dilute to 1 L 5.4 7 Potassium dihydrogen phosphate - sodium hydroxide Mix 25.0 ml of 0.2 mol/L potassium dihydrogen phosphate and 23.6 ml of 0.1 mol/L NaOH evenly, add water to dilute to 100 ml 6.8 8 Boric acid - potassium chloride - sodium hydroxide. Mix 25.0 ml 0.2 mol/L boric acid - potassium chloride and 4.0 ml 0.1 mol/L NaOH. Add water to dilute to 100 ml 8.0 9 Ammonium chloride - ammonia. Mix 0.1 mol/L ammonium chloride and 0.1 mol/L ammonia at 2: 1 Mix evenly in the ratio 9.1 10 Boric acid-potassium chloride-sodium hydroxide Mix 25.0 ml 0.2 mol/L boric acid-potassium chloride with 43.9 ml 0.1 mol/L NaOH, add water to dilute to 100 ml 10.0 11 Glycine-sodium chloride-sodium hydroxide mix 49.0 ml 0.1 mol/L Mix glycine-sodium chloride with 51.0 ml 0.1 mol/L NaOH 11.6 12 Disodium hydrogen phosphate-sodium hydroxide mix 50.0 ml 0.05 mol/L Na2HPO4 with 26.9 ml 0.1 mol/L NaOH, add water to dilute to 100 ml 12.0 13 Potassium chloride-sodium hydroxide 25.0 ml 0.2 mol/L KCl and 66.0 ml 0.2 mol/L NaOH, mix evenly, add water to dilute to 100 ml 13.0 1. Preparation of commonly used solutions (1) Precautions for solution preparation 1. Drugs must have good quality. Reagents are divided into superior pure (Guaranteed reagent, G.R.), analytical reagent (A.R.), chemical pure (C.P.), experimental reagent (Laboratory reagent, L.R.), etc. Industrial chemical reagents have many impurities and are only used in individual cases, such as sulfuric acid for washing liquids, calcium chloride for desiccants, etc. 2. Weigh medicines accurately. 3． The water for preparing reagents should be fresh deionized water or double distilled water. The specific resistance value should be above 500,000 ohms and the pH should be between 5.5 and 7.0. This requirement should be paid attention to when using tissue culture and other special purposes. Only double distilled water or deionized water is required for preparing general test solutions. 4. The prepared solution should be sterilized immediately (such as high-pressure sterilization, suction filtration or adding antibacterial substances) to prevent the growth of miscellaneous bacteria. (2) Preparation of 0.067 (1/15) Mol/L phosphate buffer 1.1/15 Mol/L potassium dihydrogen phosphate solution: Weigh 9.08g of potassium dihydrogen phosphate (KH2PO4, A.R.), dissolve it in distilled water, pour it into a 1000ml volumetric flask, and then dilute it to the mark (1000ml). 2. Preparation of 1/15Mol/L sodium dihydrogen phosphate solution: Weigh 9.47g of anhydrous disodium hydrogen phosphate (Na2HPO4, AR) (or 11.87g of Na2HPO4·2H2O), dissolve it in distilled water, put it into a 1000ml volumetric flask, and add distilled water to dilute to the mark (1000ml). 3． Prepare buffer solutions with different pH values ​​according to the proportions in the attached table. Appendix Table 1 Phosphate buffer preparation method (unit: ml) pH 1/15Mol/L Na2HPO4 1/15Mol/L KH2PO4 pH 1/15Mol/L Na2HPO4 1/15Mol/L KH2PO4 5.8 8.0 92.0 7.1 66.6 23.4 5.9 9.9 90.1 7.2 72.0 28.0 6.0 12.2 87.8 7.3 76.8 23.2 6.1 15.3 84.7 7.3 80.8 19.2 6.2 18.6 81.4 7.5 84.1 15.9 6.3 22.4 77.6 7.6 87.0 13.0 6.4 26.7 73.3 7.7 89.4 10.6 6.5 31.8 68.2 7.8 91.5 8.5 6.6 37.5 62.5 7.9 93.2 6.8 6.7 43.5 56.5 8.8 94.7 5.3 6.8 49.6 50.4 8.1 95.8 4.2 6.9 55.4 44.6 8.2 97.0 3.0 2 Preparation of commonly used pH buffer solutions and pH value 7.0 61.1 38.9 8.4 98.0 2.0 (3) 0.15Mol/L PB solution Appendix Table 2 0.15Mol/LPB liquid preparation method pH 0.15Mol/L Na2HPO4 (ml) 0.15Mol/L NaH2PO4 (ml) 6.4 26.5 73.5 6.6 37.5 62.5 6.8 49.0 51.0 7.0 61.0 39.0 7.2 72.0 28.0 7.4 81.0 19.0 7.6 87.0 13.0 Na2HPO4·2H2O molecular weight = 175.05 0.15Mol/L solution contains 26.7g/L. Na2HPO4·12H2O molecular weight = 358.22 0.15Mol/L solution contains 53.7g/L. NaH2PO4·H2O molecular weight = 138.00 0.15Mol/L solution contains 20.7g/L. NaH2PO4·2H2O molecular weight = 156.03 0.15Mol/L solution contains 23.4g/L. (4) 0.2Mol/L PB buffer Appendix Table 3 0.2mol/L PB buffer preparation method pH 0.2 Mol/L Na2HPO4 (ml) 0.2Mol/L NaH2PO4 (ml) 5.8 8.0 92.0 6.0 12.3 87.7 6.2 18.5 81.5 6.4 26.5 73.5 6.6 37.5 62.5 6.8 49.0 51.0 7.0 61.0 39.0 7.2 72.0 28.0 7.4 81.0 19.0 7.6 87.0 13.0 7.8 91.5 8.5 8.0 94.7 5.3 0.2Mol/L Na2HPO4·2 H2O solution 35.61g/L Na2HPO4·12 H2O solution contains 71.64g/L 0.2Mol/L NaH2PO4·H2O solution contains 27.60g/L NaH2PO4·2 H2O solution contains 31.21g/L If you want to prepare 0.1Mol/L PB buffer, please use 0.2Mol/L On the basis of PB buffer, add H2O to dilute 1 times. When preparing PBS solution, just add 0.85% NaCl solution to the solution. (5) Calcium- and magnesium-free phosphate buffered saline (PBS) (0.15Mol/L pH 7.2) NaCl 8.0g KCl 0.2g Na2HPO4 1.15g (2.89g if it is Na2HPO4·12H2O) KH2PO4 0.2g Dissolve the above reagents in 1 000ml of deionized water, after complete dissolution, autoclave at 115°C for 10min~15min, and store in a 4°C refrigerator for later use. This solution can be used to prepare and dilute cell dispersions and wash cell cultures. (6) Borate saline buffer Article from: Medical Park ( http://www.biodrug.cn ) tidy: zfg 1． Boric acid (H3BO3) 0.2Mol/L boric acid: Add 12.37g of boric acid to 1000ml of water. 2. Borax (Na2B4O7) 0.05Mol/L Borax: Add 19.07g of borax to 1000ml of water. Appendix Table 4 Preparation method of 0.2Mol/L boric acid buffer solution with different pH pH 0.05Mol/L borax (ml) 0.2Mol/L boric acid (ml) pH 0.05Mol/L borax (ml) 0.2Mol/L boric acid (ml) 7.4 1.0 9.0 8.2 3.5 6.5 7.6 1.5 8.5 8.4 4.5 5.5 7.8 2.0 8.0 8.7 6.0 4.0 8.0 3.0 7.0 9.0 8.0 2.0 (7) Barbiturate buffer 1. pH8.2 ionic strength 0.05Mol/L barbiturate buffer 47.6g barbiturate sodium 3 000ml distilled water 1.17N HCl 55.0ml (1.17 N HCl = 193ml concentrated HCl plus 1 807ml distilled water) ⑴ Dissolve 4.76g barbiturate sodium in 3 000ml distilled water. ⑵ Add 55ml of 1.17N HCl. ⑶ Adjust pH to 8.2 with HCl and add distilled water to 4 265ml. 2. pH8.4 0.06Mol/L barbiturate buffer, barbiturate 1.84g, barbiturate sodium 10.30g, add distilled water to 1,000.00ml 3. pH8.6 0.06Mol/L barbiturate buffer Barbiturate 1.66g Barbiturate sodium 12.76g Add distilled water 1 000.00ml (8) 0.2Mol/L acetate buffer see table Appendix 5 Appendix 5 0.2Mol/L acetate buffer preparation method pH 0.2Mol/L Tris (ml) 0.1Mol/L HCl (ml) pH 0.2Mol/L Tris (ml) 0.1Mol/L HCl (ml) 23℃ 37℃ 23℃ 37℃ 9.10 8.95 25 5 8.06 7.90 25 27.5 8.92 8.76 25 7.5 7.96 7.82 25 30.0 8.75 8.60 25 10.5 7.87 7.73 25 32.5 8.62 8.48 25 12.5 7.77 7.63 25 35.0 8.50 8.37 25 15.5 7.66 7.52 25 37.5 8.40 8.27 25 17.5 7.54 7.40 25 40.0 8.32 8.18 25 20.0 7.36 7.22 25 42.5 (9) Tris-HCl buffer (Tris-HCl buffer) Different pH, 0.05Mol/L Add хml 0.1NHCl to 25ml0.2Mol/L trishydroxymethylaminomethane, and dilute with water to 100ml (see the table below) 3 Preparation and pH value of commonly used pH buffer solutions Appendix 6 Tris-HCl buffer preparation method pH 0.2Mol/L Tris(ml) 0.1NHCl (ml) pH 0.2Mol/L Tris(ml) 0.1NHCl (ml) 23℃ 37℃ 23℃ 37℃ 9.10 8.96 25 5 8.05 7.90 25 27.5 8.92 8.78 25 7.5 7.96 7.82 25 30.0 8.74 8.60 25 10.0 7.87 7.73 25 32.5 8.62 8.48 25 12.5 7.77 7.63 25 35.0 8.50 8.37 25 15.0 7.66 7.52 25 37.5 8.40 8.27 25 17.5 7.54 7.40 25 40.0 8.32 8.18 25 20.0 7.36 7.22 25 42.5 8.23 8.10 25 22.5 7.20 7.05 25 45.0 8.14 8.00 25 25.0 25 Tris (Tris) molecular weight = 121.14 0.2Mol/L trishydroxymethylaminomethane: Tris24.23g, add water to 1000ml. 0.1NHCl: Take 8.6ml of HCl and add water to make it 1000ml. (10) 0.5Mol/L pH9.5 carbonate buffer NaHCO3 3.70g Na2CO3 0.60g Dissolve in 600.00ml distilled water. Plug the bottle tightly when not in use to avoid absorbing carbon dioxide in the air and causing the pH to drop. Best prepared in small quantities. (11) Glycerol-phosphate buffer 1. Prepare pH8.0 phosphate buffer 0.1Mol/L Na2HPO4 94.50ml 0.1N NaH2PO4 5.50ml. Mix 2.9 parts of glycerol and 1 part of pH8.0 phosphate buffer to form glycerol phosphate buffer. use: For immunofluorescence testing. (12) Hank's solution 1. Storage solution NaCl 80.00g KCl 4.00g Na2HPO4·12 H2O 1.52g KH2PO4 0.60g Glucose 4.00g 0.4% phenol red solution 50.00ml Add double distilled water to 100.00ml, autoclave at 115°C for 15 minutes, and store on ice. 2. Working fluid storage solution: 1 part double distilled water and 9 parts autoclave at 115°C for 15 minutes. Before use, use sterile 5.6% NaHCO3 solution to adjust the pH value to about 7.2 (the solution is orange-red). (13) Nessler's reagent Recipe 1: Mercury chloride 6.0g Potassium iodide 12.4g 20% ​​NaOH 30ml Add distilled water to 100ml Recipe 2: 11.5g of mercury iodide, 8g of potassium iodide, 50ml of distilled water (after complete dissolution), filter and add 50ml of 20% NaOH. use: Detect ammonium ions in solution. (14) Alsever's solution glucose 2.05g, sodium citrate 0.80g, sodium chloride 0.42g, distilled water 100ml. After dissolving, adjust the pH to 6.1 with 10% citric acid, filter, aliquot, sterilize 10 pounds for 10 minutes, and store at 4°C. (15) Penicillin and streptomycin mixture (referred to as SP solution or dual-antibody solution) Penicillin (400,000 U) 5 branched streptomycin (2 million μg) Dissolve 1 vial separately in 100 ml of sterilized deionized water or dissolve them together in 200 ml of sterilized deionized water. After mixing, divide into vials and store in a -10°C ~ -20°C refrigerator for later use (10,000 units μg/ml). Before use, add 1% of the amount into the nutrient solution or other solutions. The final concentration is 100U/ml of penicillin and 100ug/ml of streptomycin. (16) Lotion (cleaning liquid) formula 1: 150g of potassium dichromate, 300ml of distilled water, 3000ml of concentrated sulfuric acid. Add potassium dichromate to distilled water and dissolve it naturally or in a water bath. It can also be heated and dissolved in a large crucible. Then slowly add concentrated sulfuric acid and stir while adding. If the heat is excessive, stop for a moment, and then continue to add after cooling. This is a strong cleanser. The container holding the cleaning solution should be sturdy and have a thick glass lid. Wear a rubber apron, long rubber boots, glasses and thick rubber gloves when operating to ensure safety. Once the lotion turns green, it means that the chromic acid has been reduced and has lost its oxidizing ability and should not be used again. If the lotion is heated and an appropriate amount of potassium dichromate is added, it can be used again. Recipe 2: Concentrated sulfuric acid 50%, distilled water 50%, potassium dichromate 5%. This is a medium-strength cleaning solution. Recipe 3: Potassium dichromate 100g, distilled water 1000ml, heat to dissolve, cool and slowly add 4 Preparation of commonly used pH buffer solutions and pH value Industrial sulfuric acid 100ml This solution is a weak washing solution and is brown-red. When using this liquid, the glassware must be washed with hot soapy water in advance, rinsed with tap water, drained and then immersed in it, otherwise the washing liquid will lose its effectiveness quickly. 2. Preparation of calf serum Select healthy calves, generally from newborn to six months old, preferably male calves that are just born and have not yet sucked milk. They will not be fed 12 hours before blood collection and will only be given water. When collecting blood, the carotid artery, arterial cannula, latex tube, and blood container should be washed and sterilized. Put a small amount of Hank's solution into the blood collection cylinder, connect the rubber tube to the glass tube (bend) and put it into the mouth of the bottle. Then wrap the mouth of the bottle with sulfuric acid paper, white cloth and ordinary paper and tie it tightly. The intubation tube was also wrapped and tied with sulfuric acid paper. Before blood collection, the calf should be immobilized, preferably on an operating table or a fixed frame, with the head and limbs fixed, locally trimmed and sterilized, incised and stripped of the carotid artery, clamped with hemostatic forceps, sterilized, cut the artery wall, and inserted the cannula. Another assistant gently shakes the Hank's solution in the blood collection cylinder to moisten the bottle wall, which is beneficial to blood coagulation and the separation of blood clots from the bottle wall. At this time, the surgeon can bleed. The appropriate amount of blood collected in each blood collection tube should not exceed 1/3 of the total volume. If too much blood is filled, less serum will be produced. When collecting blood, you must also pay attention to the arterial cannula opening not being too thin, and the bleeding speed not being too fast. Too fast or too slow can cause hemolysis. The blood collection cartridge containing blood should be placed at room temperature (when the weather is hot) or in an incubator or greenhouse (when the weather is cold) and left to stand for more than 4 hours. After the blood has coagulated, it should be placed in a 4°C refrigerator or cold storage overnight. Then in the sterile room, carefully (do not shake) pour out or siphon out the supernatant. If mixed with red blood cells, centrifuge this part of the serum at 2 000 r/min for 10 to 15 minutes. Pay attention to aseptic operation and collect the supernatant (slightly hemolyzed serum can also be used). A calf can collect 1,500ml to 3,500ml of blood, and about half of the serum can be separated. After the serum is separated, it is inactivated in a 56°C water bath for 30 minutes to destroy complement and some infectious viruses (it does not need to be inactivated). After inactivation, it can be repackaged, and a bacteriological test is required after repackaging. If the bacterial test results are contaminated, they can be filtered with a Saishi filter. Some materials report that some serums that are not inactivated are more effective than inactivated serums. Except for the serum that will be used in the near future, which can be stored at 4°C, the rest should be stored in a low-temperature refrigerator. It is best to use the serum of 3 to 5 calves to inactivate them separately, and then mix, package and store them after bacterial testing. Mixing sera prevents individual calves from having defects that inhibit cell growth or lack certain growth factors. Article from: Medical Park ( http://www.biodrug.cn ) tidy: zfg 3. Molecular weights and equivalents of commonly used compounds Appendix Table 7 Molecular weights and equivalents of commonly used compounds Molecular formula Molecular weight equivalents Hydrochloric acid HCl 36.46 36.46 Sodium hydroxide NaOH 40.00 40.00 Potassium hydroxide KOH 56.11 56.11 Oxalic acid H2C2O4 90.04 45.02 Sodium chloride NaCl 58.44 58.44 Potassium chloride KCl 74.56 74.56 Sulfuric acid H2SO4 98.08 49.04 Acetic acid CH3COOH 60.05 60.05 Phosphoric acid H3PO4 98.00 32.67 Sodium dihydrogen phosphate NaH2PO4 119.98 Disodium hydrogen phosphate Na2HPO4 141.96 Potassium dihydrogen phosphate KH2PO4 136.09 Dipotassium hydrogen phosphate K2HPO4 174.18 Disodium diethylamine tetraacetate (EDTA—Na2) C10H14N2O8Na2 372.24 186.12 Nitric acid HNO3 63.01 63.01 Iodine I2 253.81 126.90 Potassium iodide KI 166.01 166.01 Boric acid H3BO3 61.83 20.61 Sodium bicarbonate NaHCO3 84.01 84.01 Sodium carbonate Na2CO3 105.99 53.00 Borax Na2B4O7 381.43

You are so diligent upstairs, worth learning from* .

Deionized water was used to reduce interference from other examples. The removal of carbon dioxide is to eliminate the interference of its dissolution in water on the pH.

Deionized water or distilled water should be acceptable. Remove carbon dioxide and dissolve in water to form carbonic acid, which will affect the pH value of the buffer!