Preparation HEPES Stock Solution
This guide describes the preparation of HEPES Stock Solution at a defined molarity for laboratory use.
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Stock Solution Preparation Guide – HEPES Buffer
HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid) is a zwitterionic Good’s buffer widely used in cell biology and biochemical research. It exhibits a pKa around 7.5 at 25 °C and provides effective buffering between pH 6.8 and 8.2, making it suitable for physiological pH applications. HEPES has low metal ion binding and high solubility in water.
Chemical Information
| Name | Molecular Formula | Molecular Weight | CAS |
|---|---|---|---|
| HEPES (Free Acid) | C₈H₁₈N₂O₄S | 238.30 g/mol | 7365-45-9 |
| HEPES·Na | C₈H₁₇N₂O₄S·Na | 260.29 g/mol | 75277-39-3 |
| HEPES·0.5Na | C₈H₁₇.₅N₂O₄S·0.5Na | 249.30 g/mol | 103404-87-1 |
Applications
- Biological buffer in cell culture and biochemical assays
- Maintaining physiological pH in solution
- Used in enzyme assays and electrophoresis
Preparation of 1 M HEPES Stock Solution (pH ~7.5)
Below is a typical procedure to prepare 1 M HEPES buffer stock:
- Weigh 238.30 g of HEPES (free acid).
- Add to ~800 mL of deionized water in a suitable container and begin stirring.
- Using a calibrated pH meter, slowly add strong base (e.g., 10 N NaOH) while stirring until the pH reaches the desired value (e.g., ~7.5). Monitor carefully.
- Once pH is adjusted, transfer to a 1 L graduated cylinder and add deionized water to bring the final volume to 1 L.
- Optionally filter sterilize through a 0.22 µm filter for sterile applications.
Handling and Storage
- HEPES stock solutions are clear and colorless. Store at 4 °C or room temperature for short periods.
- Prepare working buffer by diluting stock to desired concentration (e.g., 10–25 mM).
