A chelating agent is a compound that forms stable complexes with metal ions through different types of covalent or coordinate linkages during a process called chelation. Chelating agents are used during protein purification, electrophoresis, DNA extraction and several other processes.

In molecular biology, chelation is an important tool used to control metal concentrations in different experiments. Some of the ways chelating agents are used in molecular biology are to study interactions between metal-molecules, protect biological molecules from degradation by metals, or balance the metal concentrations in solutions and buffers.

This article introduces you to chelating agents and provides an overview about how chelating agents work, how they are used in molecular biology and their applications.

Article Table of Contents

How Chelating Agents Work

List of chelating agents used in molecular biology





Hexadecylpyridinium Bromide


Applications of Chelating Agents




How Chelating Agents Work

The chelating agents “catch” metals by way of their ligand binding atoms.

These ligand binding atoms are atoms that share electrons with metal ions, forming a linkage. For this reason, ligand binding atoms are also known as binding electron-donor atoms or binding atoms.

Binding atoms are a structural part of a chelating agent, and some examples include nitrogen, sulfur and oxygen (Sears, 2013).

general structure of a cheleating agent with ligand biding atoms

However, before going deeper into the mechanism of how chelating agents work, it is important to clarify that as metals are ions, they also may share electrons in the bond with the chelating agent.

Electron sharing is a foundational aspect to how chelating agents bind to metal ions where electrons can be shared in two ways: through covalent linkages and coordinate linkages.

A covalent linkage occurs when there is mutual electron sharing, meaning, both chelating agent through its binding atoms and the metal ion provides one electron each to form the bond.

general illustration of covalent bonds for chelating agents

In the case of a coordinate linkage, only the binding atoms of the chelating agent provides the two electrons to bind to the metal ion.

coordinate bonds in chelating agent illustration

Remember, to form a bond either by covalent bonding or coordinate bonding, at least two electrons must be shared (Flora and Pachauri, 2010).

But it gets a little more complex - there are a few specific ways chelating agents bind when it comes to covalent or coordinate linkages (Sears, 2013):

- One covalent and one coordinate linkage

- Two covalent linkages

- Two coordinate linkages

Finally, according to Margaret Sears’s 2013 paper, some factors influencing the successful binding between a chelating agent and a metal ion are, “the accessibility of the chelator to the tissues, how strongly the metal is already bound in the tissues, how strongly the metal binds to the chelator, and to some extent the relative quantities of various ions (Sears, 2013).”

List of chelating agents used in molecular biology

Chelating agents play several roles in molecular biology. They are used in protein purification during affinity chromatography, they can be also added to enzymes to maintain activity, they can be used during RNA and DNA extraction, and are used in several other applications.

While EDTA is a more commonly used chelating agent in many molecular applications, there are several different chelators available.

In this section, we will take a closer look at some of the different chelating agents that are used in molecular biology.


8-Hydroxyquinoline (8HQ) is a small organic molecule with chelating properties used in molecular biology, but it is also used in other fields like medicine, agriculture, biochemistry and textile industries.

This chelating agent can be derived from plants of Asteraceae and Euphorbiaceae families or produced through chemical synthesis (Prachayasittikul et al., 2013).

8HQ can form complexes with divalent metal ions (metal ions with two valence electrons), like calcium, oxygen, zinc, copper, iron, manganese, nickel and magnesium. These metals play critical functions in the metabolic equilibrium of organisms, such as cofactors in many enzymes.

8-Hydroxyquinoline (8HQ) chelating agent illustration

Other ions with which 8HQ can form complexes include toxic metals such as cadmium and aluminum (Saadeh et al., 2020).

In molecular biology, 8HQ has the following uses:

  • RNA synthesis inhibitor in yeast. 8HQ inhibits RNA polymerase activity by chelating ions like magnesium and manganese (Fraser and Creanor, 1975).
  • Chelator in cancer research studies where 8HQ and iron form a complex intercalating with DNA. This causes strong DNA damage inside cancer cells, leading to cell death.
  • Chelator in neurodegenerative disorder studies (Alzheimer's and Parkinson's diseases). 8HQ binds to Copper and Zinc ions to inhibit their linkage with amyloid-β (Aβ) peptides implicated in Alzheimer’s disease (AD) (Oliveri and Vecchio, 2021).
  • Chelator in antimicrobial activity assays. 8HQ can penetrate the bacterial cell membranes and reach metal-binding sites of bacterial enzymes, inhibiting important enzymatic activities inside bacteria (Prachayasittikul et al., 2013).
  • Inhibitor of vertebrate RNA synthesis and protein synthesis (Farrell, 2010).


Carboplatin is a synthetic chelating agent that contains platinum in its structure. The chelating reaction to carboplatin and other platinum-based molecules is called platination.

In molecular biology, carboplatin works by forming bonds with DNA. This linkage inhibits DNA replication and transcription leading to cell death (Sousa et al., 2014).


EDTA (Ethylenediaminetetraacetic acid) is a chelating agent that helps modulate enzymatic activity by controlling metal concentrations.

It is soluble in water and perfect in combination with buffers because it does not affect most chemicals present in these solutions (Lopata et al., 2019).

EDTA is commonly used in DNA preservation to inhibit the toxicity of metals which would otherwise damage DNA molecules (Sharpe et al., 2020).

Furthermore, EDTA is used in culture media to chelate calcium and reduce cell aggregation in animal cell cultures (Parzel et al., 2009).

EDTA (Ethylenediaminetetraacetic acid)


EGTA (ethylene glycol tetraacetic acid) is a calcium-specific chelator. As a result, EGTA is commonly used in applications that require calcium removal from solutions or buffers where calcium may be problematic.

Below is a list of some examples of EGTA's molecular biology applications:

  • Cell culture: EGTA is added to cell culture media to chelate calcium ions, which can be toxic to cells at high concentrations and cause cell death. By removing calcium ions, EGTA helps maintain cell viability and allows researchers to study calcium-dependent signaling pathways (Rock et al., 1997).
  • Enzyme assays: Multiple enzymes including nucleases require calcium ions as cofactors for its activity (Dominguez and War, 2009). Here, EGTA can be used to remove calcium ions from enzyme reaction buffers to study the effects of calcium on enzyme activity.
  • DNA extraction: EGTA, because of its higher affinity to calcium, is used in lysis buffers during DNA extraction to prevent degradation by calcium-dependent nucleases.
  • Protein purification: EGTA can be used in protein purification to remove calcium ions from protein samples and prevent unwanted protein-protein interactions or protein-aggregation (Khalili et al., 2004).
  • Electrophysiology: EGTA can be added to solutions to prevent calcium-dependent inactivation of voltage-gated calcium channels and improve the accuracy of electrophysiological reactions (Vargas et al., 1999).

While, EGTA and EDTA are commonly used in many applications in molecular biology, the two chelating agents differ in that EGTA has a much higher affinity for calcium than for magnesium ions compared to EDTA (Mohammadi et al., 2013).

EGTA (ethylene glycol tetraacetic acid)

Hexadecylpyridinium Bromide

Hexadecylpyridinium bromide, also known as cetylpyridinium bromide (CPB) has diverse applications in molecular biology studies including protein folding, DNA extraction, and in antimicrobial assays (Verma et al., 2015).

For instance, in protein folding, CBP can unfold and refold bovine serum albumin (BSA) by forming complexes with it. This linkage induces conformational changes in the protein, resulting in changes of polarity and stability and allows researchers to study protein functional properties (Sun et al., 2008).

In DNA extraction, CBP is used as chelating agent to induce the precipitation of DNA molecules coming from chromatography preparation (Geck and Nász, 1983). Here, hexadecylpyridinium bromide removes cations, forming cation/surfactant complexes that precipitate from an aqueous phase into an organic one (Mao et al., 2020).

Furthermore, hexadecylpyridinium bromide has been used as a bactericide in molecular biology studies. What this means is hexadecylpyridinium bromide decreases the surface tension between bacterial cell membranes and the surrounding environment through ion exchange. The interaction interrupts the osmoregulation and membrane integrity which causes cell lysis (Malek and Ramli et al., 2019).

Hexadecylpyridinium bromide, also known as cetylpyridinium bromide (CPB) illustration


Nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA) are chelating ligands commonly used for affinity chromatography and protein purification.

During affinity chromatography, NTA or IDA can be used to fix metal ions to a matrix (e.g. beads, resins). These metals can bind to proteins to enable protein purification.

To elaborate a little more, proteins can be native (unmodified) or genetically engineered containing a His-tag motif (stretch of six histidine residues added artificially to proteins on their N or C terminus).

This His-tag motif increases the affinity between protein and metals. Some of the metals used with these recombinant proteins include copper, zinc and nickel.

On the other hand, matrices such as agarose resins and beads are commonly used in affinity chromatography and agarose gels to “catch” proteins efficiently from a complex mixture.

However, naked beads cannot capture proteins, so metals are attached to these beads to form bonds with proteins. At the same time, metals cannot bind to beads by themselves. They need a bridge to connect them, and this is the role played by chelating ligands like NTA and IDA.

So, the structure formed by metal-chelating ligand-matrix is used to fix proteins to a matrix, where the protein binds by affinity to the metal.

Nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA) illustration

Applications of Chelating Agents

Metals are everywhere, in nature, in the environment, and inside our bodies, so chelators also serve purposes outside of molecular biology. Below are some different areas where chelating agents are necessary.

Geology: In geology, chelating agents can be used in chemical weathering studies. In the book Encyclopedia of Geochemistry, authors Viers and Oliva described chemical weathering as “the spontaneous and irreversible thermodynamic process that causes degradation of the mineral phases under the prevailing environmental conditions at the surface of the Earth.” (2018).

In simpler terms, the composition of mineral elements such as rocks, water and soil is constantly changing thanks to chelating reactions occurring during their interaction with the environment.

For instance, principles of chemical weathering and chelating agents are used in water treatment, as metals in pipes may cause oxidation. Here chelators like citric acid or EDTA are commonly used to capture these metals and avoid rust (Al-Qahtani, 2017).

Agriculture: Chelating agents are used in soil remediation to trap heavy metals and pollutant metals which are toxic for plant nutrition and microorganisms in soil (Nurchi et al., 2020).

For instance, heavy metals like lead and cadmium constitute a big concern in agriculture because they may cause negative health effects through food consumption (Clemens et al. 1990).

Chelating agents such as NTA and EDTA have being used to draw out metals from soil. However, according to Nurchi et al., the selection of chelators to efficiently remove pollutant metals from soil must be planned considering their physical-chemical features (e.g. solubility) (2020).

Medicine: Metal imbalance is correlated with the appearance of neurodegenerative diseases like Parkinson's and Alzheimer's (AD). Several cancers also involve enzymes with metal motifs where chelating agents are essential (Prachayasittikul et al., 2013).

Therefore, chelating agents are used in medicine to remove toxic and heavy metals from the body in what is known as chelation therapy (Flora and Pachauri, 2010).

Metals are omnipresent. They support the metabolism and homeostasis in many organisms and in the environment. Metals are not only integrated in our daily lives, but they are also a significant part of our molecular makeup. And because of that, chelating agents help during experiments to us separate metals from samples, avoid damage to biological molecules and attract metals for important molecular processes.


EDTA: Ethylenediaminotetraacetic acid

EGTA: Ethylenedioxy-diethylene-dinitrilo-tetraacetic acid

DMPS: sodium 2,3-dimercaptopropane 1-sulfonate


Chelating agent, chelators, metal ion, applications of chelating agents, examples of chelating agents.


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