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Optimizing transfer and dilution processes when using active solvent modulation in on-line two-dimensional liquid

Marie Pardon1, Soraya Chapel2, Peter de Witte3

  • 1Laboratory for Pharmaceutical Analysis, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49 box 824, 3000, Leuven, Belgium; Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Herestraat 49 box 824, 3000, Leuven, Belgium.

Analytica Chimica Acta
|March 19, 2023
PubMed
Summary
This summary is machine-generated.

Optimizing active solvent modulation (ASM) in two-dimensional liquid chromatography (2D-LC) enhances complex sample analysis. Key parameters like dilution factor and sample loop filling improve peak shape and recovery for hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC) methods.

Keywords:
Mobile phase incompatibilityOn-line dilutionSolvent strength mismatchsHILIC x RPLC

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Area of Science:

  • Analytical Chemistry
  • Chromatography

Background:

  • Two-dimensional liquid chromatography (2D-LC) is increasingly vital for complex sample analysis, driven by new commercial systems.
  • Mobile phase incompatibility between orthogonal retention mechanisms like hydrophilic interaction liquid chromatography (HILIC) and reversed-phase LC (RPLC) presents a significant challenge.
  • Active solvent modulation (ASM) offers a promising valve-based solution for on-line effluent dilution, but lacks established usage guidelines.

Purpose of the Study:

  • To investigate and optimize the active solvent modulation (ASM) process for highly incompatible LC combinations, specifically HILIC and RPLC.
  • To develop practical guidelines for utilizing ASM in simplified selective comprehensive 2D-LC (sHILIC x RPLC) setups.
  • To enhance peak shape, intensity, and recovery for early eluting compounds in complex sample analyses.

Main Methods:

  • A simplified selective comprehensive 2D-LC (sHILIC x RPLC) system was employed.
  • Key ASM parameters including dilution factor (DF), ASM phase duration, sample loop filling percentage, and unloading configuration were systematically investigated.
  • Optimization was performed using a representative complex sample to evaluate performance metrics.

Main Results:

  • Optimal conditions were identified as a dilution factor (DF) of 10, an optimized ASM phase duration, a maximum sample loop filling of 25%, and backflush mode for unloading.
  • These optimized parameters significantly improved peak shapes, intensities, and recoveries, particularly for early eluting compounds.
  • The study successfully minimized overall analysis time while maximizing chromatographic performance.

Conclusions:

  • The study provides crucial guidelines for optimizing ASM in 2D-LC, especially for HILIC x RPLC combinations.
  • The recommended parameters (DF=10, 25% loop fill, backflush unload) offer a robust strategy for managing mobile phase incompatibility.
  • These findings are applicable to various 2D-LC modes (LC x LC, LC-LC) and other chromatographic applications facing similar challenges.