Using multiple heart-cutting valves and stop-flow operation to enable variable second dimension gradient times in...

Area of Science:

• Analytical Chemistry
• Chromatography
• Separation Science

Background:

• Online comprehensive two-dimensional liquid chromatography (LC×LC) is crucial for separating complex non-volatile mixtures.
• Increasing demand for improved separation capabilities in natural products and proteomics analysis.
• Conventional LC×LC often requires very fast second dimension (²D) analyses, limiting flexibility.

Purpose of the Study:

• To develop a novel LC×LC approach enabling variable second dimension (²D) analysis times.
• To overcome the limitations of very fast ²D analyses in online LC×LC.
• To demonstrate the enhanced separation capabilities for complex natural product mixtures.

Main Methods:

• Implementation of multiple heart-cutting valves and stop-flow operation for variable ²D times.
• Application of Hydrophilic Interaction Liquid Chromatography × Reversed-Phase Liquid Chromatography (HILIC×RP-LC) for condensed tannin analysis.
• Comparison of the proposed method's performance against a conventional online LC×LC system using peak capacity.

Main Results:

• The developed method offers increased flexibility by allowing longer ²D gradients for complex chromatogram portions and shorter gradients for simpler sections.
• Demonstrated improved separation of low-level intermediate molecular weight proanthocyanidin oligomers.
• The variable gradient time stop-flow HILIC×RP-LC method showed enhanced practical peak capacities compared to conventional systems.

Conclusions:

• The proposed variable gradient time stop-flow LC×LC method provides greater flexibility and improved separation for complex mixtures.
• This approach is particularly beneficial for analyzing challenging samples like condensed tannins in natural product extracts.
• The methodology enhances the utility of online LC×LC for researchers and industry seeking higher resolution separations.