Resolving Solvent Incompatibility in Two-Dimensional Liquid Chromatography with In-Line Mixing Modulation.

Area of Science:

• Analytical Chemistry
• Chromatography Science

Background:

• Two-dimensional liquid chromatography (2D-LC) is crucial for analyzing complex samples like polymers and biomacromolecules.
• Achieving orthogonal 2D-LC separations is challenging due to incompatible chromatographic conditions.
• Existing methods like active solvent modulation (ASM) have limitations in handling complex sample matrices.

Purpose of the Study:

• To introduce and evaluate a novel in-line mixing modulation (ILMM) strategy for challenging 2D-LC workflows.
• To demonstrate the effectiveness of ILMM in coupling orthogonal chromatographic techniques.
• To compare ILMM performance against state-of-the-art active solvent modulation (ASM).

Main Methods:

• Developed and implemented an in-line mixer, termed in-line mixing modulation (ILMM).
• Applied ILMM to couple Gel Permeation Chromatography (GPC) with Reversed-Phase Liquid Chromatography (RPLC).
• Utilized ILMM for coupling Ion-Pair Reversed-Phase (IPRP) with Hydrophilic Interaction Liquid Chromatography (HILIC).
• Performed comprehensive RPLC×RPLC analysis using ILMM.

Main Results:

• ILMM significantly reduced peak distortion in GPC-RPLC, enabling a 67% higher transfer volume.
• ILMM resolved sample breakthrough issues in IPRP×HILIC for antisense oligonucleotide (ASO) analysis.
• ILMM outperformed ASM in comprehensive RPLC×RPLC analysis.
• ILMM simplified system configuration and eliminated the need for additional dilution flow.

Conclusions:

• ILMM is a facile and effective strategy for enabling challenging 2D-LC separations.
• ILMM offers significant advantages over ASM, including improved peak shape and transfer volume.
• The ILMM approach provides a versatile platform for characterizing increasingly complex molecules.