Preparation of Buffer Solutions | Phosphate, Acetate and other Buffers

Buffer solutions play an important role in ensuring product stability and providing environment for chemical and biochemical reactions. Buffers are used in analytical methods, formulation development and stability studies of products. It is essential to understand how to prepare buffer solutions correctly to achieve reliable results in laboratory and manufacturing of pharmaceutical products. This article explains the principle, preparation method, types and quality associated with buffer solutions in pharmaceutical industry.

What is a Buffer Solution?

Buffer solution is a system that resists any change in pH when a small amount of acid or base is added in the solution. It helps to maintain a consistent hydrogen concentration that is critical for reactions and product stability.
In pharmaceutical industry, buffers are used in drug formulations, biological preparations, analytical methods and culture media for microbial growth.

Expert Laboratory Guidance & Troubleshooting for Buffer Preparation

During the buffer preparation in pharmaceutical cGMP lab, it is important to accurately perform and follow the SOPs. Buffers are widely used in analysis hand have a great impact on the accuracy of today's High-Performance Liquid Chromatography (HPLC), dissolution testing and wet lab analysis. It is also important to recognize the physical and chemical variables that must be followed during preparation of buffer solutions.

Following are some technical insight into critical technical parameters and troubleshooting techniques based on industry experience to help you achieve compliance and prevent laboratory discrepancies while working in a cGMP environment.

1. Temperature Dependency of pH

A major reason for out of specification (OOS) results is due to calibrating a pH meter at one temperature, then preparing the buffer at a different temperature. Buffer solutions are temperature dependent, specifically the pH of buffer solutions is temperature dependent; therefore, it will change in value with each change in temperature (dissociation constant).

Tris Buffers: Highly temperature sensitive, if you have a Tris buffer that is prepared at pH 7.5 at 25 °C, you will get it dropped to 7.2 at 37 °C for the same buffer.
Phosphate Buffers: Moderately temperature sensitive but very temperature critical for mobility phases.

What to do: Always ensure that the final pH is measured and adjusted at the same temperature written in the particular monograph or validated analytical method (generally 20 °C or 25 °C). It is recommended to use a temperature compensated pH probe for better results.

2. Correct Sequencing to Prevent Precipitation

The order of addition of the components when making a multi-component buffer is very important when they contain volatile organic solvents or when the buffer system contains salts, such as phosphate and acetate, at very high concentrations.

The Mistake: Adding HCl or NaOH as the pH adjusting acid or base to a concentrated salt solution can create supersaturation and can lead to the precipitation of the buffer.

What to Do: Dissolve the primary buffer salt in approximately 80% of the final diluted volume of deionized / purified water (Milli-Q grade) before any of the pH adjustors (HCl or NaOH) or organic solvents (Acetonitrile or Methanol) are added, and ensure that the solutes are completely dissolved prior to adding the pH adjustors or solvents.

3. Troubleshooting Common Buffer Problems

If you've made a buffer and find out that it isn't at the target pH after you attempt to adjust it, this matrix represents some potential solutions for making corrections to your buffer while at the same time retaining the validity of your analytical results.

 

Observed Problem	Likely Technical Cause	Corrective & Preventive Action (CAPA)
pH Overshot During Adjustment	You added acid or base to the solution too quickly without stirring sufficiently to allow the buffer's capacity region to have the equilibrium change take place properly.	DO NOT "back-titrate" with the opposite reagent to the one that you used to bring the pH into range because this may change the final ionic strength of your buffer. Discard the solution and start over; when you add the adjuster, do so dropwise as you approach the target pH.
Buffer precipitated when adding the organic solvent.	The concentration of salt in the buffer after having added organic solvent exceeds the solubility of the salt in the new hydro-organic environment.	Always dilute the aqueous component of your buffer before adding organic solvent to it, or reduce the concentration of salt present in your buffer prior to adding organic solvent, provided that your validated method allows you to do so. Always add organic solvent slowly while stirring the entire solution using a magnetic stirrer.
Drifting pH Measurements	Either improper hydration of the electrode, a blockage of the reference junction or CO2 absorption from the surrounding air.	Check the slope of the electrode (should be within 95%-102%). Degas your buffer preparation water to remove dissolved CO2 and create carbonic acid that lowers your pH reading.

4. Regulatory Compliance & Document Reminders

Traceability: Record traceability of all raw materials including the manufacturer, lot number and expiry date.
Glassware Selection: Use Class A volumetric glassware for final volume adjustment, avoid storing alkaline buffers in borosilicate glass for long periods of time as silica may leach out. 

Shelf Life: Aqueous buffers (especially phosphate and acetate) must be used within 24 - 48 hours unless supported by stability studies to prevent microbial growth, which will decrease buffer capacity and interfere with chromatographic baselines.

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.

Preparation of Buffer Solutions

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°.

Related: Different Types of Titrations

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.

Related: Determination of Shelf Life of Solutions in Laboratory

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°C.

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 0.1 M hydrochloric acid and dilute with water to produce 1000 ml.

Regulatory References

1. 21 CFR Part 211.194 — Laboratory Records
2. USP Technical Repository :: General Chapter <791> pH
3. FDA Guidance Document :: ICH Q7 GMP for Active Pharmaceutical Ingredients