Popular LabKit · Recipe
Carbonate–Bicarbonate Buffer pH 9.6 (ELISA Coating Buffer)
How to prepare 0.05 M carbonate–bicarbonate buffer pH 9.6 for ELISA plate coating: grams of sodium carbonate and sodium bicarbonate for 1000 mL, and how it compares with the classic recipe.
Recipe: 0.05 M carbonate–bicarbonate buffer, pH 9.6, 1000 mL (ELISA coating)
| Component | Amount |
|---|---|
| Sodium carbonate, Na₂CO₃ (anhydrous) | 1.430 g |
| Sodium bicarbonate, NaHCO₃ | 3.067 g |
| Purified water | to 1000 mL |
The classic ELISA recipe – 1.59 g Na₂CO₃ + 2.93 g NaHCO₃ per litre – is very close: our model puts it at pH 9.66. Either recipe works; check the pH with a meter.
Other concentrations and hydrates
| Component | 0.05 M | 0.1 M |
|---|---|---|
| Sodium bicarbonate, NaHCO₃ | 3.067 g | 5.875 g |
| Sodium carbonate anhydrous, Na₂CO₃ | 1.430 g | 3.186 g |
| or sodium carbonate decahydrate, Na₂CO₃·10H₂O | 3.860 g | 8.602 g |
Method
- Weigh both salts accurately and transfer to a beaker.
- Dissolve in about 800 mL of purified water (80% of the final volume).
- Bring to the temperature of use and check the pH with a calibrated pH meter. Fine-adjust with a few drops of dilute acid or base if needed.
- Transfer to a 1000 mL volumetric flask and make up to the mark. Mix well.
- Label with name, pH, concentration, date and initials. Filter (0.22 µm) or autoclave if it must be sterile.
Make carbonate buffer fresh, or store it tightly closed for up to a week at 2–8 °C: it absorbs carbon dioxide from air and its pH slowly falls.
Recipes are calculated with the Popular LabKit buffer model (ionic strength and temperature corrected) and checked against published buffer tables. For a pharmacopoeial test, follow the reagent or buffer recipe in your monograph. Always confirm pH with a calibrated pH meter.
Frequently asked questions
Why is pH 9.6 used for ELISA coating?
At pH 9.6 most proteins carry a net negative charge and adsorb well to polystyrene plates. Use the buffer within a few days.
Can I use sodium carbonate decahydrate?
Yes. Use 2.7 times the anhydrous weight (3.860 g for 0.05 M), as in the table. Decahydrate crystals lose water over time, so the anhydrous salt gives more consistent results.
Why is the bicarbonate amount larger than the carbonate?
The second pKa of carbonic acid is about 10.3 (lower in a salt solution, about 10.0). At pH 9.6 – below the pKa – more of the buffer is in the bicarbonate form.