How to make 0.5 M EDTA, pH 8.0
The standard 0.5 M EDTA stock, with the weigh-out for the disodium dihydrate salt and an explanation of why the powder refuses to dissolve until you raise the pH to 8.
0.5 M EDTA pH 8.0 1 L of 0.5 M stock
Preparation time about 30 minutes.
| Component | Formula weight | Amount |
|---|---|---|
| EDTA disodium salt dihydrate | 372.24 g/mol | 186.1 g |
| Deionised water | — | to 1 L |
| NaOH pellets or 10 N NaOH | 40.00 g/mol | ~20 g |
Procedure
- Weigh 186.1 g of EDTA disodium dihydrate into a beaker with a large stir bar.
- Add about 800 mL of deionised water and start stirring. The powder will form a white slurry and will not dissolve — this is expected, not a mistake.
- Add NaOH slowly, either as pellets (about 20 g) or as 10 N solution, while monitoring pH. The slurry clears as the pH climbs.
- Keep adding until the pH reaches 8.0. The solution should go completely clear right around that point.
- Make up to 1 L, then autoclave or filter-sterilise.
What matters here
Why pH 8.0 is not optional
EDTA has four carboxyl groups with pKa values around 2.0, 2.7, 6.2 and 10.3. The disodium salt only becomes appreciably soluble once enough of those groups are deprotonated, which happens near pH 8. Below that it stays a slurry no matter how long you stir.
It chelates more than you intend
EDTA binds Mg²⁺, Ca²⁺, Mn²⁺, Zn²⁺ and Fe³⁺ avidly. That is the point in TE buffer (it inhibits nucleases by stripping their Mg²⁺) but it will also kill any polymerase, restriction enzyme or ligase reaction if it carries over. Keep working concentrations at or below 1 mM unless you mean it.
Sodium load
A 0.5 M stock at pH 8 carries roughly 1.4 M sodium once you count the NaOH. In low-ionic-strength applications that matters.
Frequently asked questions
Still cloudy at pH 8
Either the salt form is the free acid (which needs far more NaOH) or the pH probe is reading low. Check the bottle label for the molecular weight.
pH creeps down after autoclaving
Normal and small. Re-check and adjust if you need a tight pH.