Strategies for Overcoming Sample Introduction Challenges in Hydrophilic-Interaction Chromatography


A team of researchers has investigated strategies to overcome sample introduction challenges in hydrophilic-interaction chromatography (HILIC). By employing techniques such as pre-column dilution and selecting alternative organic solvents, they successfully addressed the issues related to injection solvent strength, resulting in improved peak symmetry and separation efficiency in HILIC analyses.

Sample introduction plays a crucial role in hydrophilic-interaction chromatography (HILIC), but it can present challenges when the injection solvent has a higher elution strength than the mobile phase (1). This can result in distorted peak shapes and reduced separation efficiency. To address this issue, researchers at the University of the West of England in Bristol investigated various strategies to manage sample introduction problems in HILIC. Their findings, published in the Journal of Chromatography A, provide valuable insights for optimizing sample injection techniques in HILIC.

Selective focus of pure acetone solution in brown glass amber bottle inside a chemistry laboratory. White background with copy space. | Image Credit: © -

Selective focus of pure acetone solution in brown glass amber bottle inside a chemistry laboratory. White background with copy space. | Image Credit: © -

One method explored by the researchers is pre-column dilution, which involves programming the injector to deliver a mixture of the weak solvent (pure acetonitrile) along with the sample dissolved in a solvent containing a higher water content than the mobile phase. This approach helps to mitigate the mismatch in elution strength between the injection solvent and the mobile phase, resulting in improved peak symmetry and separation efficiency.

Additionally, the researchers investigated the use of alternative organic solvents to acetonitrile in the injection solvent. Solvents such as isopropanol, acetone, or tetrahydrofuran were considered as potential options due to their potential to enhance sample solubility. By selecting an appropriate alternative solvent, researchers can overcome the sample introduction issues associated with using acetonitrile and achieve better chromatographic performance in HILIC.

The study also examined the impact of injection solvent volume and the potential effects of mass overload on the analysis. It was found that using smaller sample volumes is advisable to minimize any mismatch effects and improve the overall performance of the HILIC system.

David V. McCalley, the lead author of the study, emphasized the importance of managing sample introduction problems in HILIC noting that efficient sample introduction is crucial for obtaining reliable and accurate results in HILIC. By implementing suitable strategies, such as pre-column dilution and careful selection of injection solvents, researchers can overcome the challenges associated with sample injection and enhance the performance of HILIC analyses.

The findings of this research provide valuable guidance for analysts and researchers working with HILIC, offering practical approaches to address sample introduction issues. By employing these strategies, scientists can improve the accuracy, reproducibility, and efficiency of their HILIC separations, facilitating advancements in various fields such as pharmaceutical analysis, environmental monitoring, and metabolomics.


(1) Taylor, M.R.; Kawakami, J.; McCalley, D.V. Managing sample introduction problems in hydrophilic interaction liquid chromatography. J. Chromatogr. A 2023, 1700, 464006. DOI: 10.1016/j.chroma.2023.464006

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