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Modulation optimization when using a splitter pump after the first dimension in comprehensive two- dimensional liquid

Agustín Acquaviva1, Cecilia B Castells1

  • 1Laboratorio de Investigación y Desarrollo de Métodos Analíticos, LIDMA, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, 47 and 115 (B1900AJL), La Plata, Buenos Aires, Argentina; División Química Analítica, Facultad de Ciencias Exactas, UNLP, 47 and 115 (B1900AJL), La Plata, Buenos Aires, Argentina.

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Summary
This summary is machine-generated.

This study evaluates flow-splitting in online comprehensive two-dimensional liquid chromatography (LC×LC) using different pumps. Optimized setups ensure reproducible analysis of complex mixtures.

Keywords:
(2)D peak area(2)D peak dispersionActive splitter pumpLoop fillingOnline full comprehensive 2D-LCReproducibility

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Area of Science:

  • Analytical Chemistry
  • Chromatography

Background:

  • Two-dimensional liquid chromatography (2D-LC) is increasingly used for complex mixture analysis.
  • Improvements in hardware and software drive the adoption of 2D-LC.
  • Online comprehensive mode (LC×LC) offers enhanced separation power.

Purpose of the Study:

  • To evaluate the performance of online LC×LC using an active flow splitter after the first dimension.
  • To compare the effectiveness of a binary ultra-high pressure liquid chromatography (UHPLC) pump versus a syringe pump for flow splitting.
  • To analyze the impact of pump placement, tubing, and flow direction on system performance.

Main Methods:

  • Implemented an online LC×LC system with an active flow splitter post-first dimension (1D).
  • Evaluated two splitting pumps: a binary UHPLC pump and a syringe pump.
  • Assessed performance metrics including peak area reproducibility, retention time stability, and second dimension (2D) peak dispersion.
  • Investigated the influence of pump location (before/after modulation valve) and connecting tubing characteristics.
  • Analyzed the effect of flow direction on modulation valve loop filling and flushing.

Main Results:

  • Demonstrated successful flow-splitting LC×LC using both UHPLC and syringe pumps.
  • Identified flow splitting after 1D as a strategy for independent 1D/2D column selection, eluate dilution, and sample amount adaptation.
  • Showcased that system setup, particularly interface optimization, significantly impacts peak broadening and reproducibility.
  • Under optimized conditions, achieved good reproducibility in peak area and 2D dispersion, dependent on the percentage of loop filled.

Conclusions:

  • Flow-splitting is a viable and advantageous strategy for online LC×LC systems.
  • Careful optimization of the system interface and flow path is crucial for reliable and reproducible results.
  • The choice of splitting pump and its configuration can be tailored to specific analytical needs in complex mixture analysis.