How to Make a Phosphate Buffer Solution

The goal of a buffer solution is to help maintain a stable pH when a small amount of acid or base is introduced into a solution. A phosphate buffer solution is a handy buffer to have around, especially for biological applications. Because phosphoric acid has multiple dissociation constants, you can prepare phosphate buffers near any of the three pHs, which are at 2.15, 6.86, and 12.32. The buffer is most commonly prepared at pH 7 using monosodium phosphate and its conjugate base, disodium phosphate. Phosphate Buffer Materials Monosodium phosphateDisodium phosphateWaterPhosphoric acid to make the pH more acidic or sodium hydroxide to make the pH more alkalinepH meterGlasswareHot plate with stirring bar Prepare the Phosphate Buffer Decide on the concentration of the buffer. Most buffers are used at a concentration between 0.1 M and 10 M. If you make up a concentrated buffer solution, you can dilute it as needed.​Decide on the pH for your buffer. This pH should be within one pH unit from the pKa of the acid/conjugate base. So, you can prepare a buffer at pH 2 or pH 7, for example, but pH 9 would be pushing it.Use the Henderson-Hasselbach equation to calculate how much acid and base you need. You can simplify the calculation if you make 1 liter of buffer. Select the pKa value that is closest to the pH of your buffer. For example, if you want the pH of your buffer to be 7, then use the pKa of 6.9: pH pKa log ([Base]/[Acid])ratio of [Base]/[Acid] 1.096The molarity of the buffer is the sum of the molarities of the acid and conjugate base or the sum of [Acid] [Base]. For a 1 M buffer (selected to make the calculation easy), [Acid] [Base] 1.[Base] 1 - [Acid].Substitute this into the ratio and solve:[Base ] 0.523 moles/L.Now solve for [Acid]: [Base] 1 - [Acid], so [Acid] 0.477 moles/L.Prepare the solution by mixing 0.477 moles of monosodium phosphate and 0.523 moles of disodium phosphate in a little less than a liter of water.Check the pH using a pH meter and adjust the pH as necessary using phosphoric acid or sodium hydroxide.Once you have reached the desired pH, add water to bring the total volume of phosphoric acid buffer to 1 L.If you prepared this buffer as a stock solution, you can dilute it to make up buffers at other concentrations, such as 0.5 M or 0.1 M. Advantages and Disadvantages of Phosphate Buffers The two key advantages of phosphate buffers are that phosphate is highly soluble in water and that it has an extremely high buffering capacity. However, these may be offset by certain disadvantages in some situations. Phosphates inhibit enzymatic reactions.Phosphate precipitates in ethanol, so it cant be used in preparations to precipitate DNA or RNA.Phosphates sequester divalent cations (e.g., Ca2 and Mg2).