Popular LabKit · Recipe
Phosphate Buffer pH 7.4: Preparation and Recipe
How to prepare sodium or potassium phosphate buffer pH 7.4 at 0.05, 0.1 or 0.2 M. Grams of each salt for 1000 mL, for every common hydrate, with the method and the reason the amounts differ from the simple textbook formula.
Recipe: 0.1 M sodium phosphate buffer, pH 7.4, 1000 mL
| Component | Amount |
|---|---|
| Sodium dihydrogen phosphate dihydrate, NaH₂PO₄·2H₂O | 3.151 g |
| Disodium hydrogen phosphate dihydrate, Na₂HPO₄·2H₂O | 14.20 g |
| Purified water | to 1000 mL |
Using a different hydrate or concentration
Phosphate salts are sold in several hydrate forms. Use the row that matches the label on your bottle. Amounts are for 1000 mL at 25 °C.
| Salt | 0.05 M | 0.1 M | 0.2 M |
|---|---|---|---|
| NaH₂PO₄·2H₂O (dihydrate) | 1.735 g | 3.151 g | 6.244 g |
| NaH₂PO₄ (anhydrous) | 1.334 g | 2.424 g | 4.802 g |
| Na₂HPO₄·2H₂O (dihydrate) | 6.920 g | 14.20 g | 28.47 g |
| Na₂HPO₄·12H₂O (dodecahydrate) | 13.92 g | 28.58 g | 57.29 g |
| Na₂HPO₄ (anhydrous) | 5.519 g | 11.33 g | 22.71 g |
Pick one monobasic salt and one dibasic salt from the table. Mixing hydrate forms is fine as long as each weight matches its own form. At 0.2 M the ionic strength is high, so the calculation is less exact – check the pH carefully.
Potassium phosphate version
| Salt | 0.05 M | 0.1 M | 0.2 M |
|---|---|---|---|
| KH₂PO₄ (monobasic) | 1.513 g | 2.749 g | 5.447 g |
| K₂HPO₄ (dibasic, anhydrous) | 6.772 g | 13.90 g | 27.86 g |
Open the potassium phosphate version →
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.
Why these amounts?
Phosphate has a pKa of 7.20 at 25 °C, but in a real 0.1 M buffer the salts raise the ionic strength and lower the effective pKa to about 6.8. The simple Henderson–Hasselbalch equation ignores this and gives a buffer that reads about 0.2–0.4 pH units low. These recipes include the ionic-strength (Davies) correction, so they agree with the published Sigma-Aldrich phosphate tables.
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
Can I make pH 7.4 phosphate buffer from only one salt?
Yes. Dissolve the monobasic salt (e.g. KH₂PO₄ or NaH₂PO₄) at the full concentration and titrate to pH 7.4 with NaOH, using a pH meter. Pharmacopoeial pH 7.4 phosphate buffers are often made this way – follow your monograph when the method specifies it.
How long does phosphate buffer keep?
Follow your laboratory SOP – GMP laboratories commonly assign a few days to a week. Store closed, at 2–8 °C if your SOP allows, and discard at once if it turns cloudy or shows growth.
Why does my phosphate buffer pH change when I dilute it?
Diluting lowers the ionic strength, which raises the effective pKa, so the pH rises slightly (about 0.2 units for a tenfold dilution of a 0.1 M buffer). Make the buffer at the concentration you will use, or check the pH after dilution.