• Buffer theory
• Common buffers and buffer solutions
• How to prepare a buffer
• Return to Biochemistry 353

Purpose:

To prepare the following buffer solutions to be used in the purification of a-amylase and cytochrome c:
 
 

Method:

Prepare separate 0.2 M solutions of the monobasic and dibasic forms of  sodium phosphate.   Adjust the pH to 8 by  titrating the dibasic form of the buffer with the monobasic form while monitoring the pH.  Use this buffer as the stock for preparing the next two buffers.

1. Calculate and weigh out the correct amounts of Na₂HPO₄  and NaH₂PO₄  to prepare 200 mL of 0.2 M Na₂HPO₄ (dibasic) and 100 mL of NaH ₂PO₄ (monobasic), check with the instructor and prepare the solutions. (Note 1)
2. Calibrate the pH meter
3. To prepare the 0.2 M buffer, slowly add the NaH₂PO₄ (monobasic) solution to the stirred Na₂HPO₄ (dibasic) solution, until the pH = 8.0. (Note 2)
4. Use the previous buffer to prepare 1 L of 0.02 M sodium phosphate buffer, pH 8. Check the pH and adjust if necessary before diluting to the correct final volume. (Notes 1 & 2)
5. Calculate and weigh out the amount of NaCl needed to prepare 100 mL of  buffer containing 0.02 M sodium phosphate, 0.5 M NaCl , pH 8.
6. Prepare 100 mL of 0.02 M sodium phosphate, 0.5 M NaCl  buffer, pH 8, by dissolving the calculated amount of  NaCl in the appropriate amount of 0.2 M, pH 8 buffer and diluting.  Check the pH and adjust if necessary before diluting to the correct final volume. (Notes 1 & 2)
7. Check the pH of all three buffer solutions.

The pH of these three buffers must be 8 +/- 0.05 for the later experiments to work.  For best results,  prepare them carefully and don't hesitate to redo their preparation  if necessary.

Notes:

1.  Remember that molarity is based on the volume of solution, and so you should dissolve the buffer components in a volume less than the final volume, and only after the components have dissolved and the pH has been adjusted, fill to the final volume .
2.  The pH obtained in practice may differ from that calculated for a number of reasons. Some of these include;

• (a) Improperly calibrated pH meter, or pH meter calibrated at a different temperature than that of the solution measured (pH electrodes are very temperature dependent).
• (b) Wrong pK_a used, or a pK_a used that was determined at a different temperature (pK_a's are temperature dependent).
• (c) Concentration effects - ion activities, including pH, are influenced by ion concentrations. At low concentrations, ion concentration and ion activity are identical, but their values diverge with increasing ion concentration.
• (d) Wrong molecular weight used for buffer components - be sure to include the weight of any bound water in the total molecular weight of the compound. Check the side of bottle for the molecular weight of that preparation. (But see (e) and (f) below.)  
• (e) Containers for buffer components left open or poorly sealed, allowing the contents to become hydrated, or dehydrated, or absorb CO₂.   This can drastically change the molecular weight and composition of the components. Buffer components vary widely in their tendency to gain or lose bound water.  NaOH and KOH are particularly bad. 
• (f) Buffer components contaminated or partially decomposed.  
• (g) Glassware or water contaminated.

Return to top