Comprehensive two-dimensional separations based on capillary high-performance liquid chromatography and microchip...

Area of Science:

• Analytical Chemistry
• Separation Science
• Biochemistry

Background:

• Two-dimensional (2-D) separation techniques offer enhanced resolution for complex mixtures.
• Capillary liquid chromatography (LC) and microchip electrophoresis (CE) are powerful separation methods.
• Integrating these techniques can overcome limitations of individual methods.

Purpose of the Study:

• To develop and demonstrate a novel comprehensive 2-D separation system.
• To couple capillary high-performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE).
• To achieve fast sample transfers and high-efficiency separations.

Main Methods:

• A valve-free gating interface was created by inserting the LC column outlet into a specially designed chip.
• Reversed-phase cHPLC served as the first dimension, followed by chip CE as the second dimension.
• Continuous cHPLC elution with pinched injections every 20 seconds for chip CE separation, utilizing gradient elution and triethylamine buffer.

Main Results:

• The system demonstrated effective and simple comprehensive 2-D separation.
• High-sensitive on-chip detection was achieved using a home-made confocal laser-induced fluorescence detector.
• Rhodamine B showed good reproducibility with relative standard deviations of 3.2% (migration time) and 9.8% (peak height) over 150 transfers.
• Fair resolution and detection of fluorescein isothiocyanate (FITC)-labeled tryptic digests of bovine serum albumin peptides were achieved.

Conclusions:

• The developed cHPLC-chip CE system is effective for comprehensive 2-D separations.
• The valve-free interface and on-chip detection enable high-speed and sensitive analysis.
• This integrated system shows promise for analyzing complex biological samples, such as peptide digests.