Molecular and thermodynamic insights into the enthalpy-entropy shift governing HILIC retention of labelled dextrans

Grčman, Matjaž (author), Podlipnik, Črtomir (author), Pompe, Matevž (author), Kočar, Drago (author)

Hydrophilic interaction liquid chromatography (HILIC) is widely used for the analysis of glycans and oligosaccharides, yet the molecular basis of retention remains incompletely understood. In this study, we investigated dextran ladders labelled with 2-aminobenzamide (2-AB) and Rapifluor-MS™ (Waters, Milford, MA, USA) across a wide range of degrees of polymerization (DP 2–15), temperature conditions (10 °C to 70 °C), and gradient programs using a Acquity™ Premier Glycan BEH Amide column (Bridged Ethylene Hybrid, Waters, Milford, MA, USA). Van’t Hoff analysis revealed distinct enthalpic and entropic contributions to retention, allowing identification of a mechanistic transition from enthalpy-dominated docking interactions at low DP to entropy-driven dynamic adsorption at higher DP. This transition occurred reproducibly between DP 4–6, depending on the fluorescent label, while gradient steepness primarily influenced the location of the minimum enthalpy. Molecular dynamics simulations provided additional evidence, showing increased conformational flexibility and end-to-end distance variability for longer oligomers. This finding is consistent with entropy-dominated adsorption accompanied by displacement of structured interfacial water. Together, these results establish a molecular-level framework linking retention thermodynamics, conformational behavior, and solvation effects, thereby advancing our mechanistic understanding of glycan separation in HILIC.

• Language: English
• Keywords: HILIC, dextran ladder, retention mechanism, enthalpy-entropy compensation
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 12 str.
• Numbering: Vol. 30, iss. 24, art. 4711
• PID: 20.500.12556/RUL-176704
• UDC: 543.544.5
• ISSN on article: 1420-3049
• DOI: 10.3390/molecules30244711
• COBISS.SI-ID: 260633347
• Publication date in RUL: 09.12.2025

Record is a part of a journal

• Title: Molecules
• Shortened title: Molecules
• Publisher: MDPI
• ISSN: 1420-3049
• COBISS.SI-ID: 18462981

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: HILIC, dekstranska lestvica, retencijski mehanizem, entalpijsko-entropijska kompenzacija

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P1-0153-2020
• Name: Raziskave in razvoj analiznih metod in postopkov