Advancing Peptide Separations with Electrostatic Repulsion–Reversed Phase Chromatography

In this interview, Giulia Mazzoccanti from the Sapienza University of Rome discusses the development and application of electrostatic repulsion-reversed phase (ERRP) chromatography as a versatile analytical strategy for peptide separations. ERRP combines conventional reversed-phase retention with a controlled electrostatic repulsion between protonated analytes and positively charged stationary-phase sites, significantly improving selectivity, peak symmetry, and resolution. Originating from work by Guiochon and Gritti, the technique has been advanced from small basic compounds to complex glucagon-like peptide-1 (GLP-1) analogues, enabling the baseline separation of epimeric and process-related impurities that are indistinguishable by spectrometry (MS) alone. She spoke with LCGC International about her research.

You have recently published a paper detailing the performance of electrostatic repulsion-reversed phase (ERRP) chromatography approaches for the resolution of peptide mixtures (1). Please can you detail what ERRP chromatography is?

Electrostatic repulsion-reversed phase (ERRP) chromatography is a mixed-mode separation technique that combines conventional hydrophobic retention with a controlled electrostatic repulsion between protonated analytes and positively charged sites on the stationary phase.

The concept of ERRP was originally coined by Georges Guiochon and Fabrice Gritti, who demonstrated the potential of introducing positive charges onto the stationary phase to reduce secondary interactions between basic analytes and residual silanols. In their pioneering work, they employed a commercially available C18 hybrid stationary phase with a positively charged surface to apply this principle to peptides and proteins (2).

From their idea, we developed our own research path, extending the ERRP principle step by step. We first worked with small basic compounds, observing a remarkable increase in selectivity—particularly diastereoselectivity—when applying the dynamic ERRP (d-ERRP) approach on common C18 columns. This result confirmed that electrostatic repulsion not only improved peak symmetry but also enhanced stereochemical discrimination between closely related isomers (3).

Encouraged by these findings, we then moved from small molecules to peptides, beginning with glucagon and its epimeric impurity. Interestingly, the diastereoselectivity enhancement observed in small molecules was preserved in peptides: the d-ERRP system successfully resolved the glucagon epimer, maintaining the same improved selectivity trend (4).

Building upon that success, we applied the approach to icatibant and finally to liraglutide, demonstrating that both static and dynamic ERRP methods can efficiently separate peptide epimers and process-related variants (1,5,6).

This progression—from small basic compounds to complex glucagon-like peptide-1 (GLP-1) analogues—established ERRP as a powerful and versatile strategy for addressing one of the most challenging aspects of peptide analysis: the selective separation of isomeric and epimeric impurities.

In our studies, two distinct ERRP modes were explored:

• Static ERRP (s-ERRP), based on C18 hybrid stationary phase with a positively charged surface, and, for the first time, a commercially available mixed-mode hybrid C18 stationary phase with anion-exchange functionality that carries permanent positive charges in operative conditions.
• Dynamic ERRP (d-ERRP), in which the charged surface is generated in situ by continuous dynamic adsorption of a hydrophobic cation (for example, tetrabutylammonium, TBA) onto a conventional C18 column.

Thus, the ERRP principle evolved into a flexible chromatographic tool applicable to both small molecules and complex therapeutic peptides.

Can the developed ERRP separation strategies reliably differentiate between native and modified forms of GLP-1 analogues, especially in the presence of process-related impurities?

Yes—both static and dynamic ERRP have proven to be extremely effective in separating and identifying native and modified forms of GLP-1 analogues, including liraglutide, semaglutide, and exenatide (6).

These methods can achieve baseline separation of epimeric impurities such as [D-Ser]⁸, [D-His]¹, and [D-Allo-Thr]⁵, which is a remarkable achievement considering that these species are isobaric and therefore indistinguishable by mass spectrometry (MS) alone.

In practice, dynamic ERRP often delivers the highest selectivity and the sharpest, most symmetrical peaks, making it an exceptional tool for detailed impurity profiling.

At the same time, static ERRP remains the method of choice when liquid chromatography mass spectrometry (LC–MS) coupling is required; it combines excellent chromatographic performance with full MS compatibility, ensuring that separation and structural confirmation go hand in hand.

What are the main analytical challenges in characterizing epimeric impurities of GLP-1 peptides, and how does ERRP address these challenges?

Epimers are chiral molecules that differ in configuration at only one stereogenic center—a classic example is quinine and epiquinine.

Now, imagine how much more complicated things get when we move to peptides, where a long chain of amino acids may differ in the configuration of just one residue. Detecting that single change within such a complex molecule is like finding a single, subtle note out of tune in an entire symphony. To make matters worse, epimeric impurities are isobaric, meaning that even high-resolution mass spectrometry (HRMS) cannot tell them apart. They can form at different stages—during solid-phase synthesis, purification under basic conditions, or even over shelf-life storage—and they often occur at very low concentrations. Traditional reversed-phase methods struggle here: adsorption to silanol sites and peak tailing tend to hide these minor species near the main peak. This is where ERRP chromatography makes a real difference. By creating a repulsive electrostatic layer, ERRP prevents protonated peptide residues from interacting with the stationary phase surface. The result is sharper, more symmetrical peaks, higher resolution, and greater visibility of both epimeric and process-related impurities. The dynamic ERRP mode goes even further: it offers higher sensitivity, which is especially valuable when searching for trace-level impurities eluting close to the main component.

Can ERRP be used in the analysis of peptide therapeutics with similar complexity?

Yes. The whole project was developed in collaboration with the pharmaceutical company Fresenius Kabi with the aim of being universally applicable to the whole peptide class, not only to the GLP-1 agonist ones. The results obtained were pleasing and led to a versatile concept that can be successfully extended to many other therapeutic peptides. In our studies, we applied both static and dynamic ERRP to challenging molecules such as glucagon and icatibant, and the results were remarkable. We achieved excellent separation efficiency, high peak capacity, and superb peak symmetry across all systems. One of the greatest strengths of the dynamic ERRP approach is its simplicity and accessibility. Unlike static ERRP, which requires specialized columns, dynamic ERRP can be performed on any standard C18 column.

All it takes is a small tweak in the mobile phase composition, such as by simply adding a hydrophobic cation, like TBA. What makes TBA particularly convenient is that, although it behaves somewhat like a surfactant, it does not form micelles, so it doesn’t complicate the elution mechanism or alter the chromatographic mode.

Tetrabutylammonium hydrogen sulfate (TBAHSO₄) turned out to be the best-performing additive: it is highly pure, completely soluble in both water and acetonitrile, and provides a stable, reproducible electrostatic environment that enhances selectivity without complicating the method. Thanks to this straightforward setup, any laboratory equipped with a conventional C18 reversed-phase column can adopt dynamic ERRP and immediately benefit from its exceptional resolving power, with no special hardware required.

How do the different ERRP approaches (static vs. dynamic) impact the separation of GLP-1 impurities, and what are their respective advantages?

Both modes of ERRP are built on the same fundamental principle—electrostatic repulsion—but they differ in how this positive charge is created on the stationary phase.

• Static ERRP: This version uses a C18 hybrid stationary phase with a positively charged surface and a commercially available mixed-mode hybrid C18 stationary phase with anion-exchange that contains permanent positive charges on its surface. In our work, we decided to push the concept further, applying ERRP for the first time to AX/C18 columns —columns originally designed as anion exchangers. By operating them under strongly acidic conditions, we essentially turned their intended purpose upside down: the anion-exchange mechanism was suppressed, and the cationic sites of the stationary phase began to repel the protonated peptide analytes instead. This “reversed” use of AX C18 columns turned out to be a real success. It provided sharper, more symmetrical peaks, excellent reproducibility, and full MS compatibility, especially when using difluoroacetic acid (DFA) in place of TFA.
• Dynamic ERRP: Here, the positive charge isn’t built into the column; it’s generated in situ by adding TBA to the mobile phase. The TBA ions adsorb onto the surface of a standard C18 column, creating a temporary charged layer that repels protonated peptides and dramatically improves separation efficiency. The result is outstanding selectivity, beautifully symmetrical peaks, and high loadability, making it ideal for detecting trace impurities. Its only drawback is that it’s not compatible with MS detection, but for UV-based analyses, dynamic ERRP truly shines.

Can ERRP methods be integrated with mass spectrometry to provide comprehensive structural elucidation of GLP-1 impurities?

Absolutely yes for static ERRP, this is where it really proves its worth. As its stationary phases carry permanent positive charges, they are naturally compatible with LC–MS analysis.

When we use DFA instead of TFA as the mobile-phase additive, the setup becomes even more powerful: DFA maintains excellent chromatographic selectivity while greatly reducing ion suppression, allowing us to combine high-resolution separation with precise structural elucidation in a single run.

In contrast, dynamic ERRP, which relies on the adsorption of tetrabutylammonium onto a C18 surface, unfortunately, cannot be coupled with MS.

The presence of TBA causes severe ion suppression and can even contaminate the ion source, making it suitable only for UV-based analyses. For this reason, static ERRP remains the go-to approach when the goal is to integrate chromatographic resolution with LC–MS characterization—particularly in studies focused on peptide impurities and epimeric variants.

References

(1) Manetto, S.; Mazzoccanti, G.; Bassan, M.; et al. Comparing the Performance of Electrostatic Repulsion-reversed Phase Chromatography Approaches in the Resolution of Complex Peptide Mixture: Liraglutide as Case Study. Eur. J. Pharm. Sci. 2025, 211, 107120. DOI: 10.1016/j.ejps.2025.107120

(2) Gritti, F.; Guiochon, G. Separation of Peptides and Intact Proteins by Electrostatic Repulsion Reversed Phase Liquid Chromatography. J. Chromatogr. A. 2014, 1374, 112–121. DOI: 0.1016/j.chroma.2014.11.036

(3) Manetto, S.; Mazzoccanti, G.; Ciogli, A.; Villani, C.; Gasparrini, F. Ultra-high Performance Separation of Basic Compounds on Reversed-phase Columns Packed with Fully/Superficially Porous Silica and Hybrid Particles by Using Ultraviolet Transparent Hydrophobic Cationic Additives. J. Sep. Sci. 2020, 43, 1653–1662. DOI: 10.1002/jssc.201901333

(4) Mazzoccanti, G.; Manetto, S.; Bassan, M.; et al. Boosting Basic-peptide Separation Through Dynamic Electrostatic-repulsion Reversed-phase (d-ERRP) Liquid Chromatography. RSC Adv. 2020, 10, 12604–12610. DOI: 10.1039/d0ra01296c

(5) Mazzoccanti, G.; Manetto, S.; Bassan, M.; et al. Assessing the Performance of New Chromatographic Technologies for the Separation of Peptide Epimeric impurities: The Case of Icatibant. Eur. J. Pharm. Sci.2024, 193, 106682. DOI: 10.1016/j.ejps.2023.106682

(6) Mazzoccanti, G.; Manetto, S.; Bassan, M.; et al. Expanding the Use of Dynamic Electrostatic Repulsion Reversed-phase Chromatography: An Effective Elution Mode for Peptides Control and Analysis. Molecules2021, 26, 4348. DOI: 10.3390/molecules26144348

Giulia Mazzoccanti is an organic chemist and researcher at the Department of Drug Chemistry and Technologies, Sapienza University of Rome, where she earned her PhD in pharmaceutical sciences in 2018. Scientifically formed under the mentorship of Francesco Gasparrini—with whom she still collaborates—she gained expertise in chiral separations and stereochemical analysis, working first on phytocannabinoids in collaboration with Giovanni Appendino and later on peptide epimeric impurity profiling through industry-focused research with Fresenius Kabi. Her interest in chiral architectures further evolved during a visiting stay at CIC biomaGUNE (Donostia/San Sebastián, Spain) under Maurizio Prato, where she began exploring novel chiral nanomaterials for separation science and asymmetric catalysis.