The LCGC Blog: No Column to Rule Them All — Making Sense of Metal Speciation with Liquid Chromatography

Studying metals in biology is much harder than simply measuring total elemental content. Bulk techniques like atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) are excellent for quantifying how much metal is present in a sample, but they don’t tell us what that metal is actually doing (i.e., whether it’s tightly bound to a protein or loosely associated with small metabolites.³ This distinction matters because biological function and toxicity depend heavily on chemical form, not just concentration. Molecular mass spectrometry, such as electrospray ionization mass spectrometry( (ESI-MS) or matrix-assisted laser desorption (MALDI-MS), can provide structural information about metal-binding biomolecules, but it often struggles with complex mixtures and may disrupt fragile metal-ligand interactions during ionization.⁴ To access true speciation, elemental or molecular detection must be paired with separation techniques. This is where chromatography becomes critical but also complicated. Metal-binding partners span a huge size and polarity range, from large metalloproteins to small organic ligands, peptides, and inorganic complexes. No single analytical method can cleanly capture this entire landscape, which is why multiple, complementary separation strategies are often needed.

One of the more intuitive ways to tackle metal speciation is to separate complexes by size using size-exclusion chromatography (SEC). SEC distinguishes metals bound to large biomolecules, such as metalloproteins, from metals associated with low-molecular-mass ligands involved in trafficking or buffering.³ This is especially important in human biosamples, where metals may be distributed across enzymes, transport proteins, peptides, and small metabolites. Traditional protein SEC columns are optimized for tens to hundreds of kilodaltons and are great for profiling metalloproteins in serum or cerebrospinal fluid, while peptide-range SEC columns extend sensitivity down into the few-hundred dalton to kilodalton range, where many labile metal complexes reside. In practice, SEC comes with some real challenges for metal biology. First, with labile metal complexes, weakly bound metals can exchange ligands or dissociate as they move through the column, especially if mobile phase chemistry isn’t carefully controlled.⁵ Second, metals can interact with the stationary phase itself, leading to peak tailing, loss of signal, or artificial redistribution unless columns are specially treated or pre-conditioned.² Resolution is another issue: SEC can separate “big” from “small,” but it cannot distinguish between different complexes of similar size, which is common for peptides and metabolite-bound metals. As a result, SEC provides valuable size context, but rarely delivers complete speciation on its own.

One of the more recent approaches is to use hydrophilic interaction liquid chromatography, or HILIC for short. This method utilizes a mixed solvent system containing both an organic and an aqueous component with a polar stationary phase that results in separations that favor polar compounds. This method is commonly run in parallel with SEC to obtain a bigger picture, as this strategy can solve the issue of separating similarly sized species. However, it has its own shortcomings. One of the largest issues is that the approach requires a concentrated sample prior to separation, as the maximum load capacity that can be analyzed is quite low (and unfortunately, concentrated biological samples are typically hard to come by!). Additionally, each metal (and thus, metal species) will likely have different interactions with the column based on the packing material.⁶ Because of this, each metal often requires its own developed method or an entirely different HILIC column to be utilized.

Ion chromatography (IC) is one of the older approaches in our bag of metal species separation tricks. IC relies on charged groups interacting with the oppositely charged stationary phase of the column, and elution is then controlled via pH. Since many metals exist in multiple oxidation states and coordination geometries, small changes in ligand binding can shift overall charge, and thus, this approach has resulted in success for resolving and detecting many different metallocomplexes.⁷ Where this technique falls short for large and small metallocomplexes alike is the charged stationary phase. It can disrupt the interactions between the protein/small molecule and its metal, sometimes resulting in the removal of the metal entirely. Careful mobile-phase design is essential to balance resolution with preservation of native speciation.

We’d be remiss if we did not highlight reverse-phase (RP) chromatography. Commonly known for its hydrophobic separation properties, in this particular context, retention depends less on the metal itself and more on the hydrophobicity of the metallocomplex. RP lends itself to being compatible with MS due to the volatile organic mobile phases, but these acidic conditions lead to a major drawback. The binding sites for many metal complexes are typically extremely pH sensitive and thus, are easily protonated under acidic conditions.⁸ This competition with the metals for binding sites can result in the metal being displaced, altering the original complex. Nevertheless, RP chromatography has been successfully utilized to separate heme-containing metalloproteins such as hemoglobin, myoglobin, and cytochrome c.⁹

Understanding metal speciation demands analytical strategies that are just as dynamic as the metals themselves. Each chromatographic approach highlighted herein offers a different lens: SEC gives size context, HILIC emphasizes polarity, IC resolves charge differences, and RP excels for hydrophobic, kinetically stable complexes. The future of metallomics lies in integrating these tools rather than relying on any one in isolation. Multidimensional workflows and hyphenated platforms such as LC–ICP-MS coupled with molecular MS are providing simultaneous elemental and structural information under increasingly gentle conditions. At the same time, new materials, bioinert systems, and native-state separations are being developed to better preserve fragile metal-ligand equilibria. Just as important are advances in sample preparation: gentle cell lysis, metal-free buffers, anaerobic handling, and rapid fractionation strategies designed to minimize oxidation and metal redistribution in complex matrices such as blood, cerebrospinal fluid, or bacterial lysates. Because speciation can shift within minutes of disruption, preserving biological context begins long before a sample reaches the column. As these technologies and workflows mature together, our ability to map metal trafficking and dysfunction in human health will become increasingly precise and mechanistically insightful.

References
1. Afolayan, O. & Dare, E. Anatomical and Functional Distribution of Brain Metalloproteins: Roles of Iron, Copper, Zinc, and Selenium and Their Implications in Some Neurological Disorders. Niger. J. Pharm. 59, 431–445 (2025).
2. Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry | JBIC Journal of Biological Inorganic Chemistry | Springer Nature Link. https://link.springer.com/article/10.1007/s00775-021-01864-w.
3. Helle, N., Hobert, M. A., Maetzler, W. & Gledhill, M. Size-exclusion chromatography hypernated to inductively-coupled plasma and electrospray ionization mass spectrometry for the analysis of human body fluids’ metalloprotein profiles. Microchem. J. 219, 115790 (2025).
4. Brawley, H. N., Kreinbrink, A. C., Hierholzer, J. D., Vali, S. W. & Lindahl, P. A. Labile Iron Pool of Isolated Escherichia coli Cytosol Likely Includes Fe-ATP and Fe-Citrate but not Fe-Glutathione or Aqueous Fe. J. Am. Chem. Soc. 145, 2104–2117 (2023).
5. Brawley, H. N. & Lindahl, P. A. Direct Detection of the Labile Nickel Pool in Escherichia coli: New Perspectives on Labile Metal Pools. J. Am. Chem. Soc. 143, 18571–18580 (2021).
6. Köster, J., Shi, R., von Wirén, N. & Weber, G. Evaluation of different column types for the hydrophilic interaction chromatographic separation of iron-citrate and copper-histidine species from plants. J. Chromatogr. A 1218, 4934–4943 (2011).
7. Solovyev, N. et al. Biomedical copper speciation in relation to Wilson’s disease using strong anion exchange chromatography coupled to triple quadrupole inductively coupled plasma mass spectrometry. Anal. Chim. Acta 1098, 27–36 (2020).
8. Hagège, A., Huynh, T. N. S. & Hébrant, M. Separative techniques for metalloproteomics require balance between separation and perturbation. TrAC Trends Anal. Chem. 64, 64–74 (2015).
9. Aguilar, M. I. & Hearn, M. T. High-resolution reversed-phase high-performance liquid chromatography of peptides and proteins. Methods Enzymol. 270, 3–26 (1996).