0.1 M Phosphate Buffer Preparation pH 7.4
Phosphate buffer is a commonly used buffer solution in various biochemical and molecular biology applications. It is essential to prepare a phosphate buffer with a specific pH, such as pH 7.4, to maintain the optimal conditions for enzymatic reactions, protein stability, and other biological processes.
Materials
- Sodium phosphate monobasic (NaH2PO4)
- Sodium phosphate dibasic (Na2HPO4)
- Distilled water
Preparation of 0.1 M Phosphate Buffer pH 7.4
To prepare 0.1 M phosphate buffer pH 7.4, you will need to mix the two sodium phosphate salts in the correct proportions.
Step 1: Prepare the Stock Solutions
- Sodium phosphate monobasic (NaH2PO4): Weigh 12.0 grams of NaH2PO4 and dissolve it in 1000 mL of distilled water.
- Sodium phosphate dibasic (Na2HPO4): Weigh 17.9 grams of Na2HPO4 and dissolve it in 1000 mL of distilled water.
Step 2: Mix the Stock Solutions
- Mix 39.2 mL of the sodium phosphate monobasic stock solution with 60.8 mL of the sodium phosphate dibasic stock solution.
- Add distilled water to make a total volume of 1000 mL.
Step 3: Adjust the pH
- Use a pH meter to adjust the pH of the buffer solution to 7.4 by adding small amounts of either NaH2PO4 or Na2HPO4.
Step 4: Filter and Store
- Filter the buffer solution through a 0.22 μm filter to remove any impurities.
- Store the buffer solution at 4°C or freeze at -20°C for long-term storage.
Calculation
The calculation for preparing 0.1 M phosphate buffer pH 7.4 is based on the Henderson-Hasselbalch equation:
pH = pKa + log([base]/[acid])
The pKa of phosphate is 7.2. To achieve a pH of 7.4, the ratio of [base]/[acid] should be:
[base]/[acid] = 10^(pH-pKa) = 10^(7.4-7.2) = 1.58
Therefore, the ratio of Na2HPO4 to NaH2PO4 should be approximately 1.58.
Conclusion
By following these steps, you can prepare a 0.1 M phosphate buffer solution with a pH of 7.4, which is suitable for various biochemical and molecular biology applications. It is essential to maintain the correct pH and ionic strength to ensure the optimal performance of enzymes and the stability of biological molecules.
