Exploring New and Cutting-Edge LC Separation Techniques

Published on: 

At the HPLC 2023 conference in Dusseldorf, Germany, Takuya Kubo of the Graduate School of Engineering at Kyoto University in Japan presented, along with colleague Koji Otsuka, advancements in liquid chromatography the authors called "groundbreaking." Their study sheds light on the unique separation capabilities achieved through specific interactions. Notably, the researchers explored the use of π interactions and halogen-π (X-π) interactions in LC separations, as well as the separation of H/D isotopes and glycoproteins (1-8).

In previous studies, the team successfully immobilized C60-fullerene (C60) and C70-fullerene (C70) on a silica-monolithic column, uncovering the distinct characteristics of π interactions in LC (1, 2). They also investigated H/D isotope separations through CH-π interactions (3) and developed a novel monolithic column modified with corannulene (Crn) to examine multiple-pointed CH-π interactions (4).

At HPLC 2023, the researchers said they aimed to deepen their understanding of π interactions by exploring the separation of aromatic halogens, H/D isotopes, sugar chains, and glycoproteins (5). X-π interaction, a type of intermolecular interaction between halogen atoms and aromatic rings, was of particular interest.

Through comprehensive experimental evaluations, employing techniques such as UV-vis spectroscopy, 1H-NMR spectroscopy, and detailed chromatographic analyses, the researchers proposed the existence of bimodal interactions involving both π-π and X-π interactions between halogenated benzenes and aromatic materials (6). They successfully demonstrated the effective separation of di- or tri-bromo benzene isomers, various halogenated benzenes, and 11 brominated benzene analogues by optimizing the mobile phase conditions with a C70-coated column (6).

In addition to investigating halogen-π interactions, the team delved into kinetic and geometric H/D isotope effects based on OH-π and/or CH/D-π interactions in liquid chromatography (7). By leveraging the trade inquiry between kinetic and geometric isotope effects, they achieved efficient separations of H/D isotopologues, presenting a promising avenue for future research (7).

Furthermore, the researchers explored the separation of glycoproteins based on differences in their sugar chains. Traditionally, glycoprotein separation occurs under basic conditions, which may compromise the structural stability of the proteins. However, the team introduced a novel approach using 5-boronopicolinic acid (BPA), a boronic acid derivative with a lower pKa, to prepare silica-gel-based columns. These columns, packed with poly(ethylene glycol) (PEG) and poly(ethylene imine) (PEI) conjugated BPA, enabled online LC concentration of the glycoprotein HRP, even at low concentrations below the typical detection limit in LC analysis (8).

The research presented at HPLC 2023 opens up new possibilities for enhancing separation techniques in liquid chromatography. By harnessing specific interactions, scientists can overcome challenges and broaden the scope of applications in various fields, including pharmaceuticals, environmental analysis, and biotechnology.



(1) Kubo, T.; Murakami, Y.; Tsuzuki, M.; et al. Unique Separation Behavior of a C60 Fullerene-Bonded Silica Monolith Prepared by an Effective Thermal Coupling Agent. Chem – Eur. J. 2015, 21 (50), 18095–18098. DOI: 10.1002/chem.201503898

(2) Kubo, T.; Kanao, E.; Matsumoto, T.; et al. Specific Intermolecular Interactions by the Localized π-Electrons in C70-fullerene. ChemistrySelect 2016, 1 (18), 5900–5904. DOI: 10.1002/slct.201601470

(3) Kanao, E.; Kubo, T.; Naito, T.; et al. Isotope Effects on Hydrogen Bonding and CH/CD-π Interaction. J. Phys. Chem. C 2018, 122 (26), 15026–15032. DOI: 10.1021/acs.jpcc.8b04144

(4) Kanao, E.; Kubo, T.; Naito, T.; et al. Differentiating π Interactions by Constructing Concave/Convex Surfaces Using a Bucky Bowl Molecule, Corannulene in Liquid Chromatography. Anal. Chem. 2019, 91 (3), 2439–2446. DOI: 10.1021/acs.analchem.8b05260

(5) Kobayashi, H.; Okada, K.; Tokuda, S.; et al. Separation of saccharides using fullerene-bonded silica monolithic columns via π interactions in liquid chromatography. Sci. Rep. 2020, 10, 13850. DOI: 10.1038/s41598-020-70904-3

(6) Kanao, E.; Morinaga, T.; Kubo, T.; et al. Separation of halogenated benzenes enabled by investigation of halogen-π interactions with carbon materials. Chem. Sci. 2020, 11 (2), 409–418. DOI: 10.1039/C9SC04906A

(7) Kanao, E.; Kubo, T.; Naito, T.; et al. Tunable Liquid Chromatographic Separation of H/D Isotopologues Enabled by Aromatic π Interactions. Anal. Chem. 2020, 92 (5), 4065–4072. DOI: 10.1021/acs.analchem.9b05672

(8) Kobayashi, H.; Masuda, Y.; Takaya, H.; Kubo, T.; Otsuka, K. Separation of Glycoproteins Based on Sugar Chains Using Novel Stationary Phases Modified with Poly(ethylene glycol)-Conjugated Boronic-Acid Derivatives. Anal. Chem. 2022, 94 (18), 6882–6892. DOI: 10.1021/acs.analchem.2c01002