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Microchip HPLC of peptides and proteins
David S Reichmuth1, Timothy J Shepodd, Brian J Kirby
1Sandia National Laboratories, P.O. Box 969 MS 9291, Livermore, California 94551, USA.
Analytical Chemistry
|April 30, 2005
Summary
This study introduces a microchip for rapid high-performance liquid chromatography (HPLC) separations of peptides and proteins. The device enables fast, reproducible, and autonomous microscale separations, paving the way for direct mass spectrometry coupling.
Area of Science:
- Analytical Chemistry
- Microfluidics
- Biochemistry
Background:
- High-performance liquid chromatography (HPLC) is crucial for analyzing peptides and proteins.
- Miniaturization of HPLC systems offers potential for faster analyses and reduced sample consumption.
Purpose of the Study:
- To develop and demonstrate a microchip-based system for rapid reversed-phase HPLC of peptides and proteins.
- To integrate on-chip injection, separation, and detection for autonomous operation.
Main Methods:
- Utilized a microdevice with integrated subnanoliter on-chip injection and separation capabilities.
- Employed a C18 side-chain porous polymer monolith for separation and a pressure-switchable fluoropolymer valve for injection.
- Integrated a miniaturized fluorescence detector for real-time analysis.
Main Results:
- Achieved rapid separations of peptide standards and protein mixtures in 40-200 seconds.
- Demonstrated high reproducibility with a retention time RSD of 0.03% and peak area RSD of 3.8%.
- Successfully metered sample volumes from 220 to 800 pL with precise control.
Conclusions:
- The developed microchip HPLC system enables rapid, reproducible, and autonomous separations of peptides and proteins.
- The system's design facilitates direct connection to mass spectrometers at nanospray flow rates.
- This technology holds significant potential for advancing analytical workflows in proteomics and peptide analysis.