Preparation of 0.5 M EDTA Stock Solution from Anhydrous EDTA Free Acid

  • EDTA (EthyleneDiamineTetraAcetic acid), a polyamino carboxylic acid, is extensively used in molecular biology experiments as a chelating agent. It sequesters metal ions such as Zn2+, Ca2+ and Fe3+. Metal ions are necessary for the action of many enzymes including DNases.
  • EDTA is commercially available as _ _ _ _ _
    • Anhydrous EDTA (CAS Number 60-00-4, Molecular Weight 292.24)
    • Disodium EDTA dihydrate (EDTA.Na2.2H2O, CAS Number 6381-92-6, Molecular Weight 372.24) and
    • Tetrasodium EDTA tetrahydrate (EDTA.Na4.4H2O, CAS Number 13235-36-4, Molecular Weight 452.23).
  • Anhydrous free acid EDTA is least soluble among all EDTA forms in water. To dissolve anhydrous free acid EDTA in water, a lot of NaOH (3.1 ratios) is added to bring the pH to 8.0.
  • Disodium EDTA dihydrate has better solubility than anhydrous free acid EDTA and is most commonly used for the preparation of a 0.5 M EDTA solution.
  • Tetrasodium EDTA tetrahydrate is soluble in water. The resulting solution has a pH above 10.0, therefore is not suitable for cell and molecular biology experiments.
  • Here we show the preparation of 0.5M EDTA solution from Anhydrous free acid EDTA.


> EDTA (Acid free, Anhydrous)
> NaOH pallet / 10N NaOH solution (for pH adjustment)
> Deionized / Milli-Q water

Equipment and disposables
> Measuring cylinder
> Conical flask / Beaker
> Magnetic stirrer

0.5 M EDTA, pH 8.0 at 25°C

Preparation of 1000 ml of 0.5 M EDTA solution, pH 8.0 in water from Anhydrous free acid EDTA (Molecular Weight 292.24)


Step 1: Weigh out 146.12 grams EDTA.Na2.2H2O (Molecular Weight 292.24). Transfer to 2 L beaker / conical flask. Add 700 ml deionized / Milli-Q water.

Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.

Step 2: While stirring vigorously on a magnetic stirrer, add NaOH pellet to adjust the solution pH 8.0. 
Initially use NaOH pellets to adjust solution pH. When there is only a little undissolved EDTA, use 10N NaOH solution. 

EDTA dissolves slowly as you add NaOH pellets.

1. ~42 g NaOH pellet is required to adjust the pH 8.0.
2. It is not easy to dissolve EDTA. To dissolve the EDTA completely, pH 8.0 is required.

Step 3:  Adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.

Step 4:  Transfer the solution to an autoclavable bottle. Sterilize the solution by autoclaving (20 minutes at 15 lb/ (psi) from 121-124°C on liquid cycle)

Depending on the consumption, one can make small aliquots of the solution.

One can sterilize the solution by passing through a 0.22 μ filter unit. Filter sterilization removes all suspended particles with size more than 0.22 μ which includes most bacteria and their spores but not mycoplasma. However, it does not inactivate enzymatic activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.

The solution can be stored at 15 – 25 °C (room temperature) for several months.

0.5 M EDTA solution is used for the preparation of many solutions including TAE, TBE, DNA loading dye, resuspension buffer (isolation of plasmid), Tris-EDTA, Trypsin-EDTA, etc.

Follow the table to prepare an EDTA solution of specific concentration and volume from anhydrous EDTA Acid free (Molecular Weight 292.24).
Conc. / Volume100 ml250 ml500 ml1000 ml
10 mM0.29 g0.73 g1.46 g2.92 g
100 mM2.92 g7.306 g14.612 g29.224 g
0.25 M7.306 g18.265 g36.53 g73.06 g
0.5 M14.612 g36.53 g73.06 g146.12 g

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