HILIC Retention Time Issues Addressed with New Approach


Scientists from the University of Tübingen in Tübingen, Germany tested a new approach to hydrophilic-interaction chromatography (HILIC) to address retention time repeatability issues. Their findings were later published in the Journal of Chromatography A (1).

High performance liquid chromatography (HPLC) sample vial application for analysis some chemicals. The researcher placing the vial samples into the HPLC rack for auto-injection in chemistry laboratory | Image Credit: © mehmet - stock.adobe.com

High performance liquid chromatography (HPLC) sample vial application for analysis some chemicals. The researcher placing the vial samples into the HPLC rack for auto-injection in chemistry laboratory | Image Credit: © mehmet - stock.adobe.com

Reversed-phase liquid chromatography (RPLC) has become the preferred analytical technique for analyzing organic molecules in fields like pharmaceutical and food analysis. In addition to having excellent resolving power for molecules that differ in lipophilicity, it provides robust and highly repeatable separations. However, highly polar molecules like metabolites, small hydrophilic peptides and oligonucleotides are often not sufficiently retained and separated. This has led to HILIC being the favored technique for handling hydrophilic analytes. This is a normal-phase type of separation that uses reversed-phase (RP) type eluents; as such, HILIC provides a column with a hydrophilic stationary phase and an eluent with water, buffer, and a high concentration of water-miscible organic solvent (2). Part of its success is that it provides distinct and stable stationary phases with wide flexibility in the surface chemistry and selectivity. However, various studies have been reported HILIC as having poor retention time repeatability. This problem has often been ascribed to slow equilibration and insufficient re-equilibration time to establish a sensitive semi-immobilized water layer at the interface of the polar stationary phase and the bulk mobile phase.

In this study, the scientists compared retention time repeatability in HILIC for borosilicate glass and PFA (co-polymer of tetrafluoroethylene and perfluoroalkoxyethylene) solvent bottles. Borosilicate glass is a glass that contains boron trioxide, allowing for a very low coefficient of thermal expansion. This means that, unlike regular glass, it will not crack under extreme temperatures (3). Meanwhile, PFA has the advantage to withstand a higher continuous working temperature compared to FEP. Due to melt processability, PFA can be extruded in longer continuous lengths than PTFE (4). During this, they observed peak patterns shifting towards higher retention times for metabolites and peptides and lower retention times for oligonucleotides, all with ongoing analysis time when standard borosilicate glass bottles were used as solvent reservoirs. It was hypothesized that ion release (specifically from sodium, potassium, borate, and other substances) from borosilicate glass bottles leads to alterations (thickness and electrostatic screening effects) in the semi-immobilized water layer which is adsorbed to the polar stationary phase surface under acetonitrile-rich eluents in HILIC with concomitant shifts in retention.

When PFA solvent bottles were used instead of borosilicate glass, retention time repeatability was greatly improved and changed from average 8.4% relative standard deviation (RSD) for the tested metabolites with borosilicate glass bottles to 0.14% RSD for the PFA solvent bottles (30 injections over 12 h). This stems from the borosilicate glass trials leading to changes in the semi-immobilized water layer structure and thickness at the interface of the HILIC stationary phase. These effects can lead to shifted retention times, though this could be circumvented by using plastic bottles as solvent reservoirs. Similar improvements were found amongst peptides and oligonucleotides. This solution to the retention time repeatability problem in HILIC can contribute to a better acceptance of HILIC, especially in fields like targeted and untargeted metabolomics, peptide, and oligonucleotide analysis.


(1) Serafimov, K.; Knappe, C.; Li, F.; Sievers-Engler, A.; Lämmerhofer, M. Solving the Retention Time Repeatability Problem of Hydrophilic Interaction Liquid Chromatography. J. Chromatogr. A 2024, 1730, 465060. DOI: 10.1016/j.chroma.2024.465060

(2) Hydrophilic Interaction Liquid Chromatography. Merck KGaA 2024. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/analytical-chemistry/small-molecule-hplc/hilic (accessed 2024-6-25)

(3) What is Borosilicate Glass And Why Is It Better Than Regular Glass? Kablo 2023. https://shopkablo.com/blogs/the-reformist/what-is-borosilicate-glass (accessed 2024-6-26)

(4) Chemical Resistance PFA. Polyfluor Plastics bv 2023. https://www.polyfluor.nl/en/chemical-resistance/pfa/ (accessed 2024-6-26)

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