0.1 M sodium phosphate buffer, pH 7.4
Prepare the buffer from common hydrated salts.
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0.1 M sodium phosphate, pH 7.4, 1 L
at 25 °C
- Weigh 2.264 g sodium phosphate monobasic (NaH₂PO₄)
- Weigh 11.52 g sodium phosphate dibasic (Na₂HPO₄)
pKa′ 6.77 · Ionic strength 0.262 M · Buffer capacity 0.0361 M/pH
- Start with about 800 mL water.
- Add the components listed above.
- Check with a pH meter at 25 °C: adjust to pH 7.40.
- Bring to a final volume of 1 L.
Prepared by ____________________ Date __________
Buffer calculator examples
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HEPES-buffered saline, pH 7.4 at 37 °C
Adjust at room temperature before use at 37 °C.
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Mix glacial acetic acid and sodium acetate trihydrate.
Citrate buffer for antigen retrieval, pH 6.0
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Combine sodium carbonate and bicarbonate for coating.
McIlvaine citrate–phosphate buffer, pH 5.0
Mix two stocks using the published empirical table.
Glycine–HCl elution buffer, pH 2.5
Titrate glycine for an antibody elution buffer.
pH of 1× PBS without adjustment
Estimate the pH of a commonly used PBS composition.
How to make a buffer
Choose the conditions and reagents, then take the recipe to the lab.
1Choose a buffer
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2Set your conditions
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3Choose your reagents
Select the forms or stock solutions available in your lab.
4Take it to the lab
Copy or print the recipe and check pH with a meter.
Henderson–Hasselbalch equation and its corrections
Use the equation to relate pH to the acid and base forms of a buffer.
pH = pKa + log10([A−] / [HA])
pKa′ = pKa + log γA − log γHA
ln γ = −Amz²√I / (1 + 1.6√I)
ln γ = −Amz²√I / (1 + 1.6√I)
ln K(T) = ln Kr + ΔH°/R (1/Tr − 1/T) + ΔCp°/R (Tr/T − 1 + ln(T/Tr))
For 0.1 M sodium phosphate at pH 7.4, the corrected model gives pKa′ about 6.77 and about 81% dibasic phosphate.
| Method | Dibasic phosphate | Model pH |
|---|---|---|
| Ideal Henderson–Hasselbalch | 61.0% | 6.99 |
| This calculator | 81.0% | 7.40 |
| Cold Spring Harbor | 77.4% | 7.31 |
| Gomori | 81.0% | 7.40 |
References: Goldberg et al., 2002 · Cold Spring Harbor Protocols · Sørensen / Gomori
Biological buffers: pKa and temperature
Choose a buffer with pKa close to your target pH. Tris pKa rises by about 0.028 for each 1 °C decrease near room temperature.
| Buffer | pKa 25 °C | Useful pH range | dpKa/dT | pKa 4 °C | pKa 37 °C |
|---|---|---|---|---|---|
| Glycine–HCl | 2.35 | 1.35–3.35 | −0.002 /°C | 2.42 | 2.33 |
| Acetate | 4.76 | 3.76–5.76 | 2.409×10⁻⁴ /°C | 4.77 | 4.76 |
| Citrate | 3.134.766.40 | 2.13–4.133.76–5.765.40–7.40 | −0.002 /°C−0.001 /°C0.002 /°C | 3.204.826.39 | 3.114.756.43 |
| MES | 6.27 | 5.27–7.27 | −0.009 /°C | 6.47 | 6.17 |
| Bis-Tris | 6.48 | 5.48–7.48 | −0.017 /°C | 6.86 | 6.29 |
| Imidazole | 6.99 | 5.99–7.99 | −0.022 /°C | 7.48 | 6.75 |
| PIPES | 7.14 | 6.14–8.14 | −0.007 /°C | 7.29 | 7.06 |
| MOPS | 7.18 | 6.18–8.18 | −0.012 /°C | 7.46 | 7.04 |
| Sodium phosphate | 2.157.2012.35 | 1.15–3.156.20–8.2011.35–13.35 | 0.005 /°C−0.002 /°C−0.009 /°C | 2.067.2812.60 | 2.217.1812.25 |
| Potassium phosphate | 2.157.2012.35 | 1.15–3.156.20–8.2011.35–13.35 | 0.005 /°C−0.002 /°C−0.009 /°C | 2.067.2812.60 | 2.217.1812.25 |
| TES | 7.55 | 6.55–8.55 | −0.019 /°C | 7.98 | 7.33 |
| HEPES | 7.56 | 6.56–8.56 | −0.012 /°C | 7.83 | 7.42 |
| Tris | 8.07 | 7.07–9.07 | −0.028 /°C | 8.71 | 7.75 |
| Tricine | 8.13 | 7.13–9.13 | −0.018 /°C | 8.56 | 7.92 |
| Bicine | 8.33 | 7.33–9.33 | −0.015 /°C | 8.68 | 8.16 |
| Borate | 9.24 | 8.24–10.24 | −0.008 /°C | 9.45 | 9.15 |
| CHES | 9.39 | 8.39–10.39 | −0.023 /°C | 9.92 | 9.13 |
| Glycine–NaOH | 9.78 | 8.78–10.78 | −0.026 /°C | 10.37 | 9.48 |
| Carbonate–bicarbonate | 6.3510.33 | 5.35–7.359.33–11.33 | −0.005 /°C−0.009 /°C | 6.5310.56 | 6.3010.24 |
| CAPS | 10.50 | 9.50–11.50 | −0.028 /°C | 11.13 | 10.17 |
| McIlvaine citrate–phosphate | Empirical table | 2.2–8.0 | — | — | — |
References: Goldberg et al., 2002 · Good et al., 1966
Buffer calculator FAQ
How do I make 0.1 M phosphate buffer at pH 7.4?
For 1 L, use about 2.26 g sodium phosphate monobasic (NaH₂PO₄) and 11.52 g sodium phosphate dibasic (Na₂HPO₄), or about 2.60 g sodium phosphate monobasic monohydrate (NaH₂PO₄·H₂O) and 21.75 g sodium phosphate dibasic heptahydrate (Na₂HPO₄·7H₂O). Stock recipes use about 77–81 mL dibasic and 19–23 mL monobasic 1 M stock; verify with a pH meter.
Why does the Henderson–Hasselbalch equation give a different ratio?
The ideal equation treats activities as concentrations. At 0.1 M phosphate, ionic strength is about 0.26 M and pKa′ is about 6.77; the ideal 61% dibasic mixture is about pH 6.99 in the corrected model.
How much does the pH of Tris change with temperature?
Tris pKa falls by about 0.028 per °C near room temperature. A solution at pH 8.0 at 25 °C can rise by about 0.6 pH units at 4 °C; use both temperature fields to estimate the preparation reading.
How do I make Tris-HCl buffer?
Titrate Tris base (C₄H₁₁NO₃) with hydrochloric acid (HCl) and check the pH with a suitable meter. For 1 M Tris at pH 8.0, Cold Spring Harbor Protocols lists about 42 mL concentrated hydrochloric acid; this high-ionic-strength model estimates about 53 mL, so use the meter as the final guide.
What is the pH of PBS?
The listed 1× PBS composition has a calculated pH around 7.56 without adjustment and is commonly adjusted to 7.4. The 10 mM total sodium phosphate preset uses a calculated acid–base ratio, while the Cold Spring Harbor recipe uses 10 mM Na₂HPO₄ plus 1.8 mM KH₂PO₄, so the weights differ. For a fixed 10× PBS recipe, use the Molarity Calculator.
How do I make TBS or HEPES-buffered saline?
Add the appropriate sodium chloride (NaCl) concentration under Added salt. Salt changes ionic strength and therefore the estimated acid–base ratio.
What pH range does a buffer cover, and what is buffer capacity?
A useful range is approximately pKa′ ± 1 pH unit. Buffer capacity is the amount of strong base per litre needed to change pH by one unit; it generally increases with buffer concentration.
Which buffer should I use for my experiment?
Choose a buffer whose pKa′ is close to the target pH and check compatibility. Phosphate can precipitate with calcium or magnesium, Tris is temperature sensitive, and carbonate exchanges CO₂ with air.
Do the results match other buffer calculators?
For 0.1 M sodium phosphate at pH 7.4, this model gives about 81% dibasic phosphate; the Beynon calculation and the Gomori table are close. An ideal Henderson–Hasselbalch calculation gives about 61%.