0.05 M phosphate-citrate buffer (pH 5.0) recipe
The acidic buffer for dissolving HRP substrates such as TMB and OPD (McIlvaine type).
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| Na₂HPO₄ (anhydrous) | 730 mg | 51.4 mM |
| Citric acid monohydrate | 511 mg | 24.3 mM |
| Water (distilled) | to final volume |
Steps
- Dissolve in about 80% of the final volume of water.
- Check pH 5.0 and fine-tune if needed.
- Bring to the final volume with water.
- Filter and store at 4 °C.
Good to know
- By volume (100 mL): 25.7 mL 0.2 M Na₂HPO₄ + 24.3 mL 0.1 M citric acid + 50 mL water.
FAQ
How is OPD substrate made?
Add 4 mg OPD and 4 µL 30% H₂O₂ to 10 mL of this buffer just before use; read at 492 nm after stopping.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.