Comprehensive two-dimensional monolithic liquid chromatography of polar compounds

Area of Science:

• Analytical Chemistry
• Chromatography
• Separation Science

Background:

• Two-dimensional liquid chromatography (2D-LC) significantly enhances separation capabilities by combining columns with different selectivities.
• Orthogonal systems, like reversed-phase coupled with hydrophilic interaction liquid chromatography (HILIC), offer high peak capacities.
• Fast 2D-LC requires efficient micro-columns for rapid fraction transfer and high flow rate separations.

Purpose of the Study:

• To develop and evaluate novel polymethacrylate zwitterionic monolithic micro-columns for 2D-LC.
• To investigate the dual retention mechanisms (hydrophilic interaction and reversed-phase) of these monolithic columns.
• To assess the performance of on-line coupled monolithic columns for separating polar compounds.

Main Methods:

• Preparation of polymethacrylate zwitterionic monolithic micro-columns in fused silica capillaries.
• On-line coupling of a first-dimension organic polymer monolithic column with a second-dimension silica-based monolithic C18 column.
• Optimization of mobile phase composition to adjust separation selectivity for polar analytes.
• Evaluation of separation efficiency, peak capacity, and peak production rate.

Main Results:

• The developed monolithic columns exhibit dual retention mechanisms, allowing adjustable selectivity for polar compounds.
• On-line coupling of monolithic columns achieved high peak capacities in 2D-LC.
• Silica monolithic C18 columns in the second dimension demonstrated superior separation efficiency compared to particle-packed columns at high flow rates (up to 5 mL/min).
• Reducing column diameter enhanced performance by allowing higher flow rates, reducing fraction volumes, and improving mobile phase compatibility.

Conclusions:

• Polymethacrylate zwitterionic monolithic micro-columns are effective for 2D-LC, offering tunable selectivity for polar compounds.
• On-line 2D-LC systems utilizing monolithic columns provide significant improvements in resolution, peak capacity, and peak production rate.
• The use of monolithic columns, particularly silica monolithic C18 in the second dimension, is advantageous for fast and efficient separation of complex polar mixtures.