How To Make a Phosphate Buffer
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 are 2.15, 6.86 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.
1.
Decide on the Buffer Properties
Before making a buffer you must 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 L 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 correspond to that pKa.
HH Equation: pH = pKa + log ([Base]/[Acid])
For a buffer of pH 6.9, [Base]/[Acid] = 1.096
3.
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:
[Base] = 0.523 moles/L
4.
Solve for [Acid]
Using the equation: [Base] = 1 - [Acid], you can calculate that:
[Acid] = 0.477 moles/L
5.
Mix the Acid and Conjugate Base
Prepare just under 1 L of solution using the correct amounts of monosodium phosphate and
disodium phosphate.
6.
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.
7.
Correct the Volume
Once the desired pH is reached, bring the volume of buffer to 1 L.
8.
Dilute as Desired
This same buffer can be diluted to create buffers of 0.5 M, 0.1 M, 0.05 M or anything inbetween.
What You Need:

Monosodium phosphate.

Disodium phosphate.

pH meter and probe.

Phosphoric acid or sodium hydroxide to adjust pH.

Appropriate labware - volumetric flask, graduated cylinders, beakers, stir bars.

Stirring hotplate.