Phosphate Buffer Calculator (NaH2PO4 / Na2HPO4)
Calculate how much acidic and basic phosphate stock to mix for your target pH and concentration using the Henderson–Hasselbalch equation with pKa2 = 7.21 (25 °C).
What this phosphate buffer solution calculator does
This tool helps you prepare phosphate buffer by mixing two common stock solutions: sodium dihydrogen phosphate (acid form, H2PO4-) and disodium hydrogen phosphate (base form, HPO42-). You enter your desired pH, final volume, total phosphate concentration, and stock molarities, and the calculator returns the exact volume of each stock to use.
In other words, it answers the practical lab question: “How many milliliters of monobasic and dibasic phosphate do I mix?”
Core chemistry behind the calculator
Henderson–Hasselbalch equation
The phosphate buffering pair around neutral pH is:
H2PO4- ↔ H+ + HPO42-
For this pair:
pH = pKa2 + log10([base]/[acid])
where:
- base = HPO42- (typically from Na2HPO4)
- acid = H2PO4- (typically from NaH2PO4)
- pKa2 is about 7.21 at 25 °C
How volumes are obtained
Once the base/acid ratio is known from target pH, the calculator uses your requested total phosphate concentration and final volume to find total moles needed. It then splits those moles into acid and base portions and converts each portion into stock volume by dividing by stock molarity.
Example: 500 mL of 50 mM phosphate at pH 7.4
With 0.5 M stocks of both salts, a typical answer is close to:
- NaH2PO4: about 19.6 mL
- Na2HPO4: about 30.4 mL
- Water: bring to 500 mL final volume
That mix provides the required ratio for pH 7.4 while keeping total phosphate at 50 mM.
Practical lab tips for reliable phosphate buffers
1) Check temperature
pKa changes with temperature. If your work is highly pH-sensitive, adjust and verify pH at your actual working temperature.
2) Always verify pH with a calibrated meter
Calculated values are a strong starting point, but final pH should be confirmed experimentally. Calibrate your pH meter with fresh standards before final adjustment.
3) Add water last to final mark
Mix phosphate components first, then bring to final volume. This keeps concentration accurate.
4) Account for ionic strength and additives
Salts, proteins, detergents, and organic solvents can shift apparent pH. If your method includes additives, check pH after all components are present.
When phosphate buffer is a good choice
- Biochemistry workflows near neutral pH
- Protein handling and enzyme assays (when phosphate is compatible)
- General molecular biology prep
- Cell-free systems requiring stable pH around 6.5–8.0
Limitations and compatibility notes
- Phosphate can precipitate with divalent cations (e.g., Ca2+, Mg2+) under some conditions.
- Not ideal for every enzyme or metal-dependent system.
- For extremes of pH, other buffers may be more appropriate.
Quick FAQ
Can I use this for PBS preparation?
Yes, this is directly relevant for the phosphate part of PBS. Just account for NaCl, KCl, and other salts separately.
Why does the calculator warn me that water volume is negative?
That means your target concentration is too high for the stock molarities and final volume selected. Use more concentrated stocks or lower the target phosphate concentration.
Should I trust exact decimal places?
Use the result as a precise guide, but always make final pH adjustments experimentally.