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Preparation of Buffer Solutions

Learn how to prepare different types of buffer solutions like phosphate buffer solution, ammonia buffers, ammonium buffers, acetate buffers and citrate buffers from USP, BP and IP used in chemical analysis of Pharmaceutical ingredients.

Standard Buffer Solutions

Standard Buffer Solutions are solutions of standard pH. They are used for reference purposes in pH measurements and for carrying out many pharmacopoeial tests which require adjustments to or maintenance of a specified pH. They may be prepared by the methods described below. The preparation of special buffer solutions is described in the sections in which their use is specified as in the microbiological assay of antibiotics or in the individual monographs where the use of such solutions is indicated.

The reagents required for the preparation of standard buffer solutions are described here. All the crystalline reagents except boric acid should be dried at 110° to 120°C for 1 hour before use. Carbon dioxide-free water should be used for preparing buffer solutions and wherever water is mentioned for preparation of such solutions the use of carbon dioxide-free water is implied.

The prepared solutions should be stored in chemically resistant, glass-stoppered bottles of alkakli-free glass and used within 3 months of preparation. Any solution which has become cloudy or shows any other evidence of deterioration should be discarded.

Standard buffer solutions for various ranges of pH values 1.2 to 10.0 may be prepared by appropriate combinations of 0.2 M hydrochloric acid or 0.2 M sodium hydroxide and of solutions described below, used in the proportions shown in the accompanying tables. The standard pH values given in the tables and elsewhere in the Appendix are considered to be reproducible within ± 0.02 unit at 25°.
1. Boric Acid and Potassium Chloride, 0.2 M: Dissolve 12.366 g of boric acid and 14.911 g of potassium chloride in water and dilute with water to 1000 ml.
2. Disodium Hydrogen Phosphate, 0.2 M: Dissolve 71.630 g of disodium hydrogen phosphate in water and dilute with water to 1000 ml.
3. Hydrochloric Acid, 0.2 M: Hydrochloric acid diluted with water to contain 7.292 g of HCl in 1000 ml.
4. Potassium Chloride, 0.2 M: Dissolve 14.911 g of potassium chloride in water and dilute with water to 1000 ml.
5. Potassium Dihydrogen Phosphate, 0.2 M: Dissolve 27.218 g of potassium dihydrogen phosphate in water and dilute with water to 1000 ml.
6. Potassium Hydrogen Phthalate, 0.2 M: Dissolve 40.846 g of potassium hydrogen phthalate in water and dilute with water to 1000 ml.
7. Sodium Hydroxide, 0.2 M: Dissolve sodium hydroxide in water to produce a 40 to 60 percent w/v solution and allow to stand. Taking precautions to avoid absorption of carbon dioxide, siphon off the clear supernatant liquid and dilute with carbon dioxide-free water, a suitable volume of the liquid to contain 8.0 g of NaOH in 1000 ml.
NOTE - 0.2 M Sodium hydroxide must not be used later than one month after preparation.

The composition of Standard Buffer Solutions

Hydrochloric Acid Buffer: Place 50 ml of the 0.2M potassium chloride in a 200 ml volumetric flask, add the specified volume of 0.2 M hydrochloric acid (see Table I) and then add water to volume.
Acid Phthalate Buffer: Place 50.0 ml of 0.2 M potassium hydrogen phthalate in a 200 ml volumetric flask, add the specified volume of 0.2 M hydrochloric acid (see Table 2) and then add water to volume.
Neutralized Phthalate Buffer; Phthalate Buffer: Place 50.0 ml of 0.2 M potassium hydrogen phthalate in a 200 ml volumetric flask, add the specified volume of 0.2 M sodium hydroxide (see Table 3) and then add water to volume.
Phosphate Buffer Solution Preparation: Place 50.0 ml of 0.2 M potassium dihydrogen phosphate in a 200 ml volumetric flask, add the specified volume of 0.2 M sodium hydroxide (see Table 4) and then add water to volume.
Alkaline Borate Buffer: Place 50.0 ml of 0.2 M boric acid and potassium chloride in a 200 ml volumetric flask, add the specified volume of 0.2 M sodium hydroxide (see Table 5) and then add water to volume.

Other Buffer Solutions

Acetate Buffer pH 2.8: Dissolve 4 g of anhydrous sodium acetate in about 840 ml of water, add sufficient glacial acetic acid to adjust the pH to 2.8 (about 155 ml) and dilute with water to 1000 ml.
Acetate Buffer pH 3.4: Mix 50 ml of 0.1 M sodium acetate with 950 ml of 0.1 M acetic acid.
Acetate Buffer pH 3.5: Dissolve 25 g of ammonium acetate in 25 ml of water and add 38 ml of 7 M hydrochloric acid. Adjust the pH to 3.5 with either 2 M hydrochloric acid or 6 M ammonia and dilute with water to 100 ml.
Acetate Buffer pH 3.7: Dissolve 10 g of anhydrous sodium acetate in 300 ml of water, adjust the pH to 3.7 with glacial acetic acid and dilute with water to 1000 ml. Before use adjust to pH 3.7, if necessary, with glacial acetic acid or anhydrous sodium acetate, as required.
Acetate Buffer pH 4.0: Place 2.86 ml of glacial acetic acid and 1.0 ml of a 50 percent w/v solution of sodium hydroxide in a 1000 ml volumetric flask, add water to volume and mix. Adjust the pH, if necessary.
Acetate Buffer pH 4.4: Dissolve 136 g of sodium acetate and 77 g of ammonium acetate in water and dilute with water to 1000 ml. Add 250 ml of glacial acetic acid and mix.
Acetate Buffer pH 4.6: Dissolve 5.4 g of sodium acetate in 50 ml of water, add 2.4 ml of glacial acetic acid and dilute with water to 100 ml. Adjust the pH, if necessary.
Acetate Buffer pH 4.7: Dissolve 8.4 g of sodium acetate and 3.35 ml of glacial acetic acid in sufficient water to produce 1000 ml. Adjust the pH, if necessary.
Acetate Buffer pH 5.0: Dissolve 13.6 g of sodium acetate and 6 ml of glacial acetic acid in sufficient water to produce 1000 ml. Adjust the pH, if necessary.
Acetate Buffer pH 5.5: Dissolve 272 g of sodium acetate in 500 ml of water by heating to 35°, cool and add slowly 50 ml of glacial acetic acid and sufficient water to produce 1000 ml. Adjust the pH, if necessary.
Acetate Buffer pH 6.0: Dissolve 100 g of ammonium acetate in 300 ml of water, add 4.1 ml of glacial acetic acid, adjust the pH, if necessary, using 10M ammonia or 5 M acetic acid and dilute with water to 500 ml.
Acetate Buffer Solution: Dissolve 14 g of potassium acetate and 20.5 ml of glacial acetic acid in sufficient water to produce 1000 ml.
Acetate-Edetate Buffer pH 5.5: Dissolve 250 g of ammonium acetate and 15 g of disodium edetate in 400 ml of water and add 125 ml of glacial acetic acid in sufficient water to produce 1000ml.
Acetic Acid-Ammonium Acetate Buffer: Dissolve 77.1 g of ammonium acetate in water, add 57 ml of glacial acetic acid and dilute with water to 1000 ml.
Acetic Ammonia Buffer pH 3.7, Ethanolic: To 15 ml of 5 M acetic acid add 60 ml of ethanol (95 percent) and 24 ml of water. Adjust the pH to 3.7 with 10 M ammonia and dilute with water to 100 ml.
Acetone Solution, Buffered: Dissolve 8. 15 g of sodium acetate and 42 g of sodium chloride in water, add 68 ml of 0.1 M hydrochloric acid and 150 ml of acetone and dilute with water to 500 ml.
Albumin Phosphate Buffer Solution pH 7.2; Phosphate-albumin
Buffered Saline pH 7.2: Dissolve 10.75 g of disodium hydrogen phosphate, 7.6 g of sodium chloride and 10 g of bovine albumin in sufficient water to produce 1000 ml. Before use adjust to pH 7.2 with 2 M sodium hydroxide or a 10 percent w/v solution of phosphoric acid as required.
Ammonia-Ammonium Chloride Buffer: Dissolve 67.5 g of ammonium chloride in about 200 ml of water, add 570 ml of strong ammonia solution and dilute with water to 1000 ml.
Ammonia Buffer pH 9.5: Dissolve 33.5 g of ammonium chloride in ISO ml of water, and 42 ml of 10M ammonia and dilute with water to 250 ml. Store in polyethylene containers.
Ammonia Buffer pH 10.0: Dissolve 5.4 g of ammonium chloride in 20 ml of water, add 35 ml of 10 M ammonia and dilute with water to 100 ml.
Ammonia Buffer pH 10.9: Dissolve 67.5 g of ammonium chloride in sufficient 10 M ammonia to produce 1000 ml.
Barbitone Buffer pH 7.4: Mix 50 ml of the solution containing 1.944 percent w/v of sodium acetate and 2.946 percent w/v of barbitone sodium with 50.5 ml of 0.1 M hydrochloric acid, add 20 ml of an 8.5 percent w/v solution of sodium chloride and dilute with water to 250 ml.
Barbitone Buffer pH 8.6, Mixed; Barbitone Buffer pH 8.6: Dissolve 1.38 g of barbitone, 8.76 g of barbitone sodium and 0.38 g of calcium lactate in sufficient water to produce 1000 ml.

Boric Buffer pH 9.0; Borate Buffer pH 9.0: Dissolve 6.20 g of boric acid in 500 ml of water, adjustto pH 9.0 with] M sodium hydroxide (about 41.5 ml) and dilute with water to 1000 ml.
Buffer Solution pH 2.5: To 25.0 ml of 0.2 M potassium hydrogen phthalate add 37.0 ml of 0.1 M hydrochloric acid and dilute with sufficient water to produce 100.0 ml.
Buffer (HEPES) solution pH 7.5: Dissolve 2.38 g of 2[4-( hydroxyethyl)piperazin-1ethanesulphonic acid in about 90 ml of water. Adjust the pH to 7.5 with sodium hydroxide solution. Dilute to 100 ml with water.
Carbonate Buffer pH 9.7: Dissolve 8.4 g of sodium bicarbonate and 10.6 g of sodium carbonate in sufficient water to produce 500 ml.
Chloride Buffer pH 2.0: Dissolve 6.57 g of potassium chloride in water, add 119.0 ml of 0.1 M hydrochloric acid and dilute with water to 1000 ml.
Citrate Buffer: Dissolve 0.5 g of citric acid monohydrate and 0.4 g of dibasic sodium phosphate in sufficient water to produce 1000 ml.
Citro-phosphate Buffer pH 5.0: Mix 48.5 ml of 0.1 M citric acid with sufficient 0.2 M disodium hydrogen phosphate to produce 100 ml.
Citro-phosphate Buffer pH 6.0: Mix 36.8 ml of a 2.1 percent w/v solution of citric acid with 63.2 ml of a 7.15 percent w/v solution of disodium hydrogen phosphate.
Citro-phosphate Buffer pH 7.0: Mix 17.6 ml of a 2.1 percent w/v solution of citric acid with 82.4 ml of a 7.15 percent w/v solution of disodium hydrogen phosphate.
Citro-phosphate Buffer pH 7.2: Mix 13.0 ml of a 2.1 percent w/v solution of citric acid with 87.0 ml of a 7.15 percent w/v solution of disodium hydrogen phosphate.
Citro-phosphate Buffer Solution pH 7.6: Dissolve 1.33 g of citric acid and 67.1 g of disodium hydrogen phosphate in sufficient water to produce 1000 ml.
Cupric Sulphate Solution pH 2.0, Buffered: Mix 5.3 ml of 0.2 M hydrochloric acid and 25 ml of 0.2 M potassium chloride, add 4 ml of a 0.393 percent w/v solution of cupric sulfate and dilute to 100ml of water.
Cupric Sulphate Solution pH 4.0, Buffered: Dissolve 0.25 g cupric sulfate and 4.5 g of ammonium acetate in sufficient water to produce 100 ml.
Cupric Sulphate Solution pH 5.2, Buffered: Dissolve 1.522 g of anhydrous disodium hydrogen phosphate in sufficient water to produce 53.6 ml and add a 2.1 percent solution of citric acid until the pH of the solution is between 5.15 and 5.25 (about 46 ml). Mix 98.5 ml of the resulting solution with 1.5 ml of a 0.393 percent solution of cupric sulfate.
Diethanolamine Buffer pH 10.0: Dissolve 96.4 g of diethanolamine in sufficient water to produce 400 ml. Add 0.5 ml of an 18.6 percent w/v solution of magnesium chloride, adjust the pH to 10.0 with 1 M hydrochloric acid and dilute with water to 500 ml.
Glycine Buffer pH 11.3: Mix a solution containing 0.75 percent w/v of glycine and 0.58 percent w/v of sodium chloride with an equal volume of 0.1 M sodium hydroxide. Adjust the pH if necessary.
Glycine Buffer Solution: Mix 42 g of sodium bicarbonate and 50 g of potassium bicarbonate with 180 ml of water and add a solution containing 37.5 g of glycine and IS ml of strong ammonia in 180 ml of water. Dilute with water to 500 ml and stir until solution is complete.
Imidazole Buffer pH 6.5: Dissolve 6.81 g of imidazole and 1.23 g of magnesium sulfate in 752 ml of 0.1 M hydrochloric acid, adjust the pH if necessary and dilute with water to produce 1000 ml.
Imidazole Buffer pH 7.4: Dissolve 3.40 g of imidazole and 5.84 g of sodium chloride in water, and 18.6 ml of 1 M hydrochloric acid and dilute with water to produce 1000 ml.
Palladium Chloride Solution, Buffered: To 0.5 g of palladium chloride add 5 ml of hydrochloric acid and warm on a water bath. Add 200 ml of hot water in small portions with continued heating until the solution is complete. Cool and dilute with sufficient water to produce 250.0 ml. To 50.0 ml of the resulting solution add 10.0 ml of 1 M sodium acetate, 9.6 ml of 1 M hydrochloric acid and sufficient water to produce 100.0 ml.

Phosphate-albumin buffered saline pH 7.2: Dissolve 10.75 g of disodium hydrogen phosphate, 7.6 g of sodium chloride and 10 g of bovine albumin in water and dilute to 1000.0 ml with the same solvent. Immediately before use adjust the pH using dilute sodium hydrogen solution or dilute phosphoric acid.
Phosphate Buffer pH 2.0: Dissolve 0.136 g of potassium dihydrogen phosphate in 800 ml of water, adjust the pH to 2.0 with hydrochloric acid and add sufficient water to produce l000ml.
Phosphate Buffer pH 2.5: Dissolve 100 g of potassium dihydrogen phosphate in 800 ml of water, adjust the pH to 2.5 with hydrochloric acid and add sufficient water to produce l000ml.
Phosphate Buffer pH 3.0: Dissolve 1.36 g of potassium dihydrogen orthophosphate and 2 ml of triethylamine in 800 ml of water, adjust the pH to 3.0 with orthophosphoric acid and add sufficient water to produce 1000 ml.
Phosphate Buffer pH 3.6: Dissolve 0.900 g of anhydrous disodium hydrogen phosphate and 1.298 g of citric acid monohydrate in sufficient water to produce 1000 ml.
Phosphate Buffer pH 4.0, Mixed: Dissolve 5.04 g disodium hydrogen phosphate and 3.01 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml. Adjust the pH with glacial acetic acid.
Phosphate Buffer pH 4.9: Dissolve 40 g of sodium dihydrogen phosphate and 1.2 g of sodium hydroxide in sufficient water to produce 100 ml. If necessary, adjust the pH with 1 M sulphuric acid or 1 M sodium hydroxide as required.
Phosphate Buffer pH 5.0: Dissolve 6.8 g of potassium dihydrogen phosphate in 1000 ml of water and adjust the pH to 5.0 with ]0 M potassium hydroxide.
Phosphate Buffer pH 5.5, Mixed:
SOLUTION I - Dissolve 13.61 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml.
SOLUTION II - Dissolve 35.81 g of disodium hydrogen phosphate in sufficient water to produce 1000 ml.
Mix 96.4 ml of solution I with 3.6 ml of solution II.
Phosphate Buffer pH 6.5: Dissolve 60.5 g of disodium hydrogen phosphate and 46 g of potassium dihydrogen phosphate in water, add 100 ml of 0.02 M disodium edentate and 20 mg of mercuric chloride and dilute with water to produce I000 ml.
Phosphate Buffer pH 6.8, Mixed: Dissolve 28.20 g of disodium hydrogen phosphate and 11.45 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml.
Phosphate Buffer pH 6.8, 0.2 M Mixed: Dissolve 13.872 g of potassium dihydrogen phosphate and 35.084 g of disodium hydrogen phosphate in sufficient water to produce 1000 ml. Store in a cold place.
Phosphate Buffer pH 7.0, Mixed: Dissolve 0.50 g of anhydrous disodium hydrogen phosphate 0.301 g of potassium dihydrogen phosphate in sufficient water to produce
Phosphate Buffer pH 7.0 with Azide, Mixed: To 1000 ml of a solution containing 1.8 percent w/v of disodium hydrogen phosphate and 2.3 percent w/v of sodium chloride, add sufficient of a solution containing 0.78 percent w/v of sodium dihydrogen phosphate and 2.3 percent w/v of sodium chloride (about 280 ml) to produce a pH of 7.0. Dissolve sufficient sodium azide in the resulting solution to give a 0.02 percent w/v solution.
Phosphate Buffer pH 7.0, 0.067 M Mixed: Dissolve 3.532 g of potassium dihydrogen phosphate and 14.542 g of disodium hydrogen phosphate in sufficient water to produce 1000 ml.
Phosphate Buffer pH 7.0, 0.1 M Mixed; Phosphate Buffer pH 7.0, 0.1 M: Dissolve 1.361 g of potassium dihydrogen orthophosphate in sufficient water to produce 100 ml and adjust the pH using 3.5 percent w/v solution of disodium hydrogen orthophosphate.
Phosphate Buffer pH 7.5: Dissolve 6.8 g of potassium dihydrogen orthophosphate and 1.56 g of sodium hydroxide in 900 ml of water adjust the pH 7.5 with sodium hydroxide solution and dilute with water to produce 1000 ml.
Phosphate Buffer pH 7.5, 0.2 M: Dissolve 27.2 g of potassium dihydrogen phosphate with 930 ml of water adjust the pH 7.5 with 0.3 percent w/v solution of potassium hydroxide and add sufficient water to produce 1000 ml.
Phosphate Buffer pH 7.5, 0.33 M Mixed:
SOLUTION I - Dissolve 119.3 I g of disodium hydrogen phosphate in sufficient water to produce 1000 ml.
SOLUTION II - Dissolve 45.36 g of potassium dihydrogen phosphate in sufficient water to produce 1000 ml.
Mix 85 ml of solution I and 15 ml of solution II and adjust the pH if necessary.
Phosphate Buffer pH 8.0, 0.02 M: Mix 50 ml of 0.2 M potassium dihydrogen phosphate with 46.8 ml of 0.2 M sodium hydroxide and add sufficient water to produce 500 ml.
Phosphate Buffer, 0.025 M Standard: Dissolve 3.40 g of potassium dihydrogen phosphate and 3.55 g of anhydrous disodium hydrogen phosphate, both previously dried at 110° to 130° for 2 hours, in sufficient water to produce 1000 ml.
Phosphate Buffer, 0.05 M: Dissolve 6.8 g of potassium dihydrogen orthophosphate in sufficient water to produce l000ml.
Saline, Phosphate-buffered: Dissolve 2.5 g of sodium dihydrogen phosphate, 2.523 g of disodium hydrogen phosphate and 8.2 g of sodium chloride in sufficient water to produce 1000 ml.
Saline pH 6.4, Phosphate-buffered: Dissolve 1.79g of disodium hydrogen phosphate, 1.36 g of potassium dihydrogen phosphate and 7.02 g of sodium chloride in sufficient water to produce 1000 ml.
Saline pH 7.4, Phosphate-buffered: Dissolve 2.38 g of disodium hydrogen phosphate, 0.19 g of potassium dihydrogen phosphate and 8.0 g of sodium chloride in sufficient water to produce 1000 ml. Adjust the pH, if necessary.

Tris-Acetate Buffer pH 8.5: Dissolve 0.294 g of calcium chloride and 12.11 g of tris (hydroxymethyl) aminomethane in water. Adjust the pH with 5 M acetic acid and dilute to 1000.0 ml with water.
Tris-Chloride Buffer pH 7.4: Dissolve 7.27 g of tris (hydroxymethyl) methylamine and 5.27 g of sodium chloride adjust the pH, if necessary and dilute with water to produce 1000 ml.
Tris (hydroxymethyl) aminomethane Buffer pH 7.4: Dissolve 30.3 g of tris (hydroxymethyl) aminimethane in approximately 200 ml of water. Add 183 ml of I M hydrochloric acid. Dilute to 500.0 ml with water.
NOTE - The pH is 7.7-7.8 at room temperature and 7.4 at 37°. This solution is stable for several months at 4°.
Tris (hydroxymethyl) aminomethane Buffer pH 8.1: Dissolve 2.9 g of calcium chloride with 400 ml of tris (hydroxymethyl) aminomethane solution adjust the pH with] M hydrochloric acid and dilute with water to produce l000 ml.

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.

2. As for the acetate buffers: Are we talking anhydrous or mono-, tri or tetrahydrate sodium acetate?

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