EDTA and chelating agents: how they bind metal ions in lab solutions

EDTA (ethylenediaminetetraacetic acid) is a chelating agent: one molecule wraps around a single metal ion and holds it through six binding sites. It is one of the most common additives in a lab. It turns up in buffers, enzyme work, cleaning solutions and classic titrations. Its job is always the same: it grips metal ions tightly and stops them taking part in other reactions. This guide explains how EDTA and other chelating agents work, why they are added to solutions, how EDTA titrations measure metals, and when EDTA causes problems, including with a metal–peptide complex such as GHK-Cu.

Terms are defined in the glossary.

What a chelating agent is

A chelating agent is a molecule that binds a metal ion through two or more atoms at once. The name comes from the Greek for a crab's claw: the molecule closes around the metal from several sides.

Binding through several atoms makes a much more stable complex than binding through one. Each attachment point helps hold the others in place, so the metal is hard to pull away. This extra stability is called the chelate effect.

The number of atoms a molecule uses to bind is its denticity:

What EDTA is

EDTA stands for ethylenediaminetetraacetic acid. Its structure is a short two-carbon bridge with a nitrogen at each end, and two acetic acid arms on each nitrogen. That gives six atoms that can bind a metal: the two nitrogens and one oxygen from each of the four carboxyl groups.

EDTA binds almost every metal ion with a charge of +2 or more, always one EDTA to one metal. How tightly depends on the metal. Published binding constants (log K, the higher the tighter), measured at 20 °C, give a sense of the range (Harvey, Analytical Chemistry 2.1):

Each step of 1 in log K means ten times tighter binding. So EDTA holds copper more than 100 million times more tightly than it holds calcium.

The forms you buy

• EDTA free acid dissolves very poorly in water.
• Disodium EDTA dihydrate (Na₂H₂EDTA·2H₂O, 372.24 g/mol) is the usual lab form. Even this dissolves slowly until the pH is raised to about 8 with sodium hydroxide. A 0.5 M stock at pH 8.0 is a standard recipe.
• Tetrasodium EDTA dissolves easily but gives a strongly alkaline solution.

Making a 0.5 M EDTA stock at pH 8

1. Work out the mass. Disodium EDTA dihydrate has a molar mass of 372.24 g/mol (PubChem), so 1 litre of 0.5 M needs 0.5 × 372.24 = 186.1 g. For 100 mL, weigh 18.61 g.
2. Add it to about 80% of the final volume of purified water and stir. It won't dissolve yet.
3. Raise the pH with sodium hydroxide, added slowly while stirring and watching a calibrated pH meter. The solid dissolves as the pH approaches 8.
4. Set the pH to 8.0, then make up to the final volume in a volumetric flask.

For the arithmetic behind step 1, see molarity and dilutions.

Why pH matters

EDTA's carboxyl and amine groups must lose their protons to bind a metal. At low pH, hydrogen ions compete for those groups, and EDTA binds metals less well. Most EDTA work is therefore done at neutral to alkaline pH. For how pH controls which groups carry a proton, see buffers and pH.

Why EDTA is added to buffers

Limiting metal-catalysed oxidation

Water, salts and glassware all carry traces of iron and copper. These ions are catalysts: they speed up the reaction between oxygen and sensitive groups such as cysteine thiols and methionine, without being used up (Stadtman, Free Radic. Biol. Med. 1990). A small amount of EDTA, often around 1 mM, ties these ions up so they can't take part.

This also protects reducing agents. DTT is oxidised by trace metals, and was found to last longer in buffers that contain a metal-binding agent (Getz et al., Anal. Biochem. 1999). For more on oxidation and reducing agents, see oxidation and reduction.

Stopping metal-dependent enzymes

Many enzymes need a metal ion to work, often magnesium, calcium or zinc. Adding EDTA removes the metal and stops the enzyme. Labs use this to:

• stop an enzyme reaction at a set time,
• protect samples from metal-dependent proteases, enzymes that cut peptide bonds, and
• keep DNA buffers, such as Tris-EDTA, free of enzymes that need magnesium.

Other common uses

• Cleaning. EDTA solutions remove metal deposits and scale from glassware and equipment.
• Water treatment. EDTA ties up calcium and magnesium, the ions behind hard water.

EDTA titrations

Because EDTA binds metals one-to-one, it can be used to measure how much metal a solution contains. This is a complexometric titration.

1. Buffer the sample, for example to pH 10 with ammonia buffer.
2. Add an indicator dye that binds the metal and changes colour when it lets go. Eriochrome Black T is a classic choice for calcium and magnesium, used between about pH 7.5 and 10.5 (Harvey, Analytical Chemistry 2.1). It is wine red when bound to the metal and blue when free. It works like a pH indicator, but responds to a metal ion instead of a proton; see pH indicators.
3. Add EDTA solution of known concentration from a burette. EDTA takes the metal from the dye because it binds more tightly.
4. Stop at the colour change. When all the metal is bound to EDTA, the dye turns blue. The volume of EDTA used gives the amount of metal.

The best-known example is measuring water hardness, the total of calcium and magnesium. For another type of titration used on COAs, see Karl Fischer titration.

When EDTA gets in the way

EDTA removes metals that a method needs, not just the ones it doesn't. Leave it out, or remove it first, when the work depends on a metal:

EDTA and GHK-Cu

GHK-Cu is a coordination complex: the tripeptide GHK holds a copper(II) ion through three nitrogen atoms, with water or another oxygen donor in the fourth position. See GHK-Cu: the chemistry of a peptide–copper complex.

EDTA holds copper(II) through six atoms and binds it very tightly. In a solution containing both, EDTA can take copper away from GHK. The blue colour of the complex fades as copper moves to EDTA, whose copper complex absorbs differently. So buffers that contain EDTA or another strong metal-binding agent aren't suitable for work that needs the copper complex intact.

Other chelating agents

Handling EDTA

EDTA salts are stable white powders. Store them dry at room temperature, and make up stock solutions in purified water, since tap water's calcium and magnesium would use up some of the EDTA. Check the safety data sheet for the form you use. See COSHH and safety data sheets.

Related guides

For the bonds that hold metal complexes together, see chemical bonds and polarity. For the groups in a peptide that can bind metals, see functional groups in peptides. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.