How to Make a Phosphate Buffer

These buffers can be used for biological applications at near neutral pH

What makes phosphate buffers stand out? Firstly, because it contains three acidic protons, phosphoric acid has multiple dissociation constants and can be used to create buffers for either of the three corresponding pHs.

The three pKa values for phosphoric acid (from the CRC Handbook of Chemistry and Physics) are 2.16, 7.21 and 12.32. Monosodium phosphate and its conjugate base, disodium phosphate are usually used to generate buffers of pH values around 7, for biological applications, as shown here.

Making this buffer is a bit harder than making TAE and TBE buffers. It is not extremely hard, however. Simply average. It should take about 10 minutes to whip up this buffer. Here's how.

1. Decide on the Buffer Properties

Before making a buffer, you should first know what molarity you want it to be, what volume to make and what the desired pH is. Most buffers work best at concentrations between 0.1 M and 10 M. The pH should be within 1 pH unit of the acid/conjugate base pKa. For simplicity, this sample calculation will be for 1 liter of buffer.

2. Determine the Ratio of Acid to Base

Use the Henderson-Hasselbalch equation (below) to determine what ratio of acid to base is required to make a buffer of the desired pH. Use the pKa value nearest your desired pH and the ratio will refer to the acid-base conjugate pair that corresponds to that pKa.

HH Equation: pH = pKa + log ([Base]/[Acid])

For a buffer of pH 6.9, [Base]/[Acid] = 0.4898

Substitute for [Acid] and Solve for [Base]

The desired molarity of the buffer is the sum of [Acid] + [Base].

For a 1 M buffer, [Base] + [Acid] = 1 and [Base] = 1 - [Acid]

By substituting this into the ratio equation, from step 2, you get:

[Acid] = 0.6712 moles/L

Solve for [Acid]

Using the equation: [Base] = 1 - [Acid], you can calculate that:

[Base] = 0.3288 moles/L

3. Mix the Acid and Conjugate Base

After you've used the Henderson-Hasselbalch equation to calculate what ratio of acid to base is required for your buffer, prepare just under 1 liter of solution using the correct amounts of monosodium phosphate and disodium phosphate.

4. Check the pH

Use a pH probe to confirm that the correct pH for the buffer is reached. Adjust slightly as necessary, using phosphoric acid or sodium hydroxide (NaOH).

5. Correct the Volume

Once the desired pH is reached, bring the volume of buffer to 1 liter. Then dilute the buffer as desired. This same buffer can be diluted to create buffers of 0.5 M, 0.1 M, 0.05 M or anything in between.

6. Tips for the Buffer

Remember that pKa is not easily measured to an exact value. Slightly different values might be available in the literature from different sources.

7. What You Need

To make the buffer, you'll need three substances, while the remaining materials are equipment. The substances are monosodium phosphate, disodium phosphate, and phosphoric acid or sodium hydroxide to adjust pH.

You'll also need a pH meter and probe and appropriate labware. This includes a volumetric flask, graduated cylinders, beakers and stir bars.

Lastly, you'll need a stirring hotplate.