Materials:
1. Deionized water
2. Potassium dihydrogen phosphate (KH2PO4)
3. Sodium hydrogen phosphate (Na2HPO4)
4. pH meter
5. Magnetic stirrer or stir bar
6. Volumetric flask (e.g., 1000 mL)
7. Graduated cylinder or pipette
Reagents:
1. 0.2 M Potassium dihydrogen phosphate (KH2PO4) solution: Dissolve 17.41 g of KH2PO4 in 1000 mL of deionized water.
2. 0.2 M Sodium hydrogen phosphate (Na2HPO4) solution: Dissolve 28.48 g of Na2HPO4 in 1000 mL of deionized water.
Procedure:
1. Calculate the volumes of 0.2 M KH2PO4 and 0.2 M Na2HPO4 solutions needed to prepare 1000 mL of 0.1 M phosphate buffer at pH 7.5. Use the Henderson-Hasselbalch equation:
pH = pKa + log ([A-]/[HA])
where:
- pH is the desired pH (7.5)
- pKa is the dissociation constant of phosphoric acid (7.21)
- [A-] is the concentration of the conjugate base (Na2HPO4)
- [HA] is the concentration of the weak acid (KH2PO4)
Rearranging the equation and substituting the values:
log ([A-]/[HA]) = pH - pKa = 7.5 - 7.21 = 0.29
[A-]/[HA] = antilog (0.29) = 1.959
From the desired buffer concentration (0.1 M) and the calculated ratio ([A-]/[HA]), determine the volumes of 0.2 M KH2PO4 and 0.2 M Na2HPO4 solutions:
V (KH2PO4) = [HA] / [Total Concentration] * Total Volume
= 0.1 M / (0.1 M + 0.1 M) * 1000 mL
= 500 mL
V (Na2HPO4) = [A-] / [Total Concentration] * Total Volume
= 0.1 M / (0.1 M + 0.1 M) * 1000 mL
= 500 mL
2. Transfer 500 mL of 0.2 M KH2PO4 solution into a 1000 mL volumetric flask.
3. Add 500 mL of 0.2 M Na2HPO4 solution to the volumetric flask.
4. Swirl the flask gently to mix the solutions.
5. Check the pH of the buffer using a pH meter. Adjust the pH to 7.5 by adding small volumes of either 0.2 M KH2PO4 or 0.2 M Na2HPO4 solutions as needed.
6. If necessary, add deionized water to bring the final volume of the buffer up to 1000 mL.
7. Mix the buffer thoroughly using a magnetic stirrer or stir bar.
Your 0.1 M phosphate buffer of pH 7.5 is now ready to use. Store the buffer at the appropriate temperature for your experimental conditions.
