On-column quantification of amino functionalities bonded to solid porous matrices packed within high performance...

Area of Science:

• Analytical Chemistry
• Chromatography
• Materials Science

Background:

• Silica-based stationary phases (SPs) functionalized with amino groups are crucial for Hydrophilic Interaction Liquid Chromatography (HILIC) separations.
• These SPs effectively resolve diverse biologically relevant compounds like carbohydrates and nucleosides.
• Quantifying surface functionalization is key to understanding and optimizing SP performance.

Purpose of the Study:

• To modify and validate a procedure for quantifying amino groups on silica matrices packed within High-Performance Liquid Chromatography (HPLC) columns.
• To enable in-depth analysis of the structural composition of functionalized stationary phases.
• To assess the impact of functionalization on column performance and reusability.

Main Methods:

• Adaptation of the non-destructive DNase I-mediated binding assay (DNBA-M) procedure for HPLC columns.
• Validation of the modified OC-DNBA-M procedure using aminopropyl-silica matrices.
• Analysis of SPs with varying degrees of ureidic functionalization.

Main Results:

• The OC-DNBA-M procedure was successfully validated for quantifying amino groups on silica SPs inside HPLC columns.
• The method provided in-depth structural analysis of SPs, including those with partial ureidic functionalization.
• Analyzed columns retained their chromatographic performance after the analysis, demonstrating non-destructive application.

Conclusions:

• The OC-DNBA-M procedure offers a reliable and non-destructive method for characterizing amino-functionalized silica SPs in HPLC columns.
• This technique facilitates a deeper understanding of SP structure-retention relationships in HILIC.
• The validated method supports the development and optimization of advanced chromatographic materials.