Saturday, May 9, 2009

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.
Time Required: 10 minutes

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 in-between.

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.


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