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Reversed HILIC Gradient: A Powerful Strategy for On-Line Comprehensive 2D-LC
Soraya Chapel1,2, Florent Rouvière1, Davy Guillarme3,4
1Institut Des Sciences Analytiques, Université de Lyon, UMR 5280, CNRS, 5 rue de la Doua, 69100 Villeurbanne, France.
Molecules (Basel, Switzerland)
|May 13, 2023
Summary
Reversed hydrophilic interaction chromatography (revHILIC) offers unique selectivity for analyzing peptides and pharmaceuticals. This technique, especially in two-dimensional liquid chromatography (2D-LC), provides complementary separation compared to traditional methods.
Area of Science:
- Analytical Chemistry
- Chromatography
- Separation Science
Background:
- Hydrophilic interaction chromatography (HILIC) is a common technique for separating polar compounds.
- Reversed hydrophilic interaction chromatography (revHILIC) utilizes a polar stationary phase with a reversed acetonitrile gradient.
- Two-dimensional liquid chromatography (2D-LC) enhances separation power by combining orthogonal chromatographic techniques.
Purpose of the Study:
- To evaluate the capabilities and constraints of the reversed hydrophilic interaction chromatography (revHILIC) mode in liquid chromatography (LC).
- To compare the retention behavior of revHILIC with reversed-phase LC (RPLC) and HILIC.
- To assess the performance of revHILIC in comprehensive two-dimensional LC (LC × LC) configurations.
Main Methods:
- Comparison of retention behavior using representative mixtures of peptides and pharmaceutical compounds across RPLC, revHILIC, and HILIC.
- Evaluation of two-dimensional LC (2D-LC) conditions by coupling RPLC, revHILIC, and HILIC with RPLC in an on-line comprehensive (LC × LC) mode.
- Analysis of selectivity, peak capacity, and sensitivity for different LC × LC combinations.
Main Results:
- Selectivity order was determined as RPLC > revHILIC > HILIC for both peptide and pharmaceutical mixtures.
- revHILIC coupled with RPLC in a 2D-LC setup (revHILIC × RPLC) demonstrated high peak capacity and sensitivity.
- revHILIC × RPLC provided complementary selectivity to RPLC × RPLC and HILIC × RPLC, with significant differences in elution order and retention time.
Conclusions:
- revHILIC offers distinct selectivity advantages over traditional HILIC and can be a valuable addition to RPLC.
- revHILIC is a viable and powerful option for comprehensive 2D-LC (LC × LC) analyses.
- The revHILIC × RPLC combination is particularly effective for the 2D-LC analysis of small molecules and peptides, offering improved separation and sensitivity.
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