Multidimensional Liquid Chromatography Covered at HTC 2024


As part of the 18th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology, held May 28–31, 2024 in Leuven, Belgium, a workshop session titled “Multidimensional Liquid Chromatography” was held on May 30. Led by Session Chair Oliver J. Schmitz of the University Duisburg-Essen in Germany, this session focused on the various ways multidimensional liquid chromatography (LC) can be used in different applications.

View on medieval St Peter's church and traditional brick houses in Leuven, Belgium | Image Credit: © SvetlanaSF -

View on medieval St Peter's church and traditional brick houses in Leuven, Belgium | Image Credit: © SvetlanaSF -

First, André de Villiers of Stellenbosch University in South Africa presented “Detailed investigation of cannabis phenolics using 1- and 2-dimensional liquid chromatography hyphenated to ion mobility spectrometry and high-resolution MS.” The cannabis industry has grown significantly in recent years, and while cannabis phytochemistry, particularly regarding cannabinoid and terpenoid composition, has been extensively studied, little emphasis has been placed on the phenolic composition of cannabis. Phenolics can affect cannabis in various ways, with the plant potentially being a source of valuable flavonoids. However, less than 50 flavonoids have been reported in cannabis to date, with little information available regarding their variation between strains. In this study, 13 cannabis strains were grown with phenolic extracts of collected plant material being analyzed with 1-dimensional reversed phase LC (RP-LC) hyphenated to cyclic ion mobility spectrometry (cIMS)-high resolution MS (HR-MS), as well as comprehensive two-dimensional hydrophilic interaction chromatography (HILIC) × RP-LC-HR-MS.

Following this talk, Pia Wittenhofer of the University Duisburg-Essen in Germany presented “Heart-Cut 2D LC-MS as Tool for the Analysis of Cholesterol Biosynthesis in Pancreatic and Melanoma Cancer.” In recent studies, cholesterol has been shown to play a role in various types of cancers, showing the importance of understanding corresponding biosynthetic pathways. However, characterizing cholesterol biosynthesis poses different analytical challenges, which stem from structural similarities, matrix interferences, and significant concentration differences among precursor molecules and cholesterol itself. For this study, a novel heart-cut two-dimensional liquid chromatography–mass spectrometry (2D LC–MS) method was designed to address these challenges.

The next presentation, “Overcoming the challenge of large-volume modulations in coupling organic SEC with RPLC to empower forensic characterization of explosives,” was presented by Rick S. van den Hurk of Universiteit van Amsterdam in The Netherlands. One of the biggest challenges surrounding 2D LC–MS is solvent incompatibility. This occurs when two complementary separation modes are coupled where the solvent of the first dimension (1D) is a strong eluting solvent for the second-dimension (2D) separation mode. Different strategies have been used to address this, but some of these methods are not optimal in extreme scenarios where large modulation volumes are needed to maintain sensitivity in heart-cut 2D–LC applications. For this study, the scientists combined size-exclusion chromatography (SEC) and reversed-phase liquid chromatography (RPLC) in an online heart-cut 2D–LC configuration to address this issue.

Following this, Oskar Munk Kronik of the University of Copenhagen presented “An in-depth evaluation of the advantages and limitations of HILIC, RPLC, RPLC×HILIC, and HILIC×RPLC coupled to UV (- cyclic IMS) - HRMS for phenolic and flavonoid analysis in non-target screening.” Multidimensional LC and ion mobility spectrometry (IMS) have become more popular due to a desire to better resolve isomers and compounds of interest in complex samples. Reversed phase liquid chromatography (RPLC) and hydrophilic interaction chromatography (HILIC) coupled to ultraviolet (UV) and high-resolution mass spectrometry (HRMS) detection is often used for phenolic and flavonoid analysis. Here, the scientists presented these analytical properties in different combinations to show their advantages and limitations.

Finally, Marie Pardon of KU Leuven in Belgium presented “A detailed comparison of in-house developed and commercial two-dimensional liquid chromatography systems in heart-cutting and selective comprehensive modes.” 2D–LC is becoming popular for analyzing complex samples, especially with the introduction of commercial 2D–LC systems. In this study, commercial and in-house developed 2D–LC systems were compared based on experimental differences, cost, flexibility, and more.


(1) 5A: Multidimensional Liquid Chromatography. Dr. H. Weinreich, Hamburg, Germany 2024. (accessed 2024-5-30)

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