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Updated: Jun 24, 2026
10:05
Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry
Published on: October 24, 2018
Comprehensive two dimensional separation based on coupling micellar electrokinetic chromatography with capillary
1Department of Chemistry, University of Waterloo, ON, Canada.
The Analyst
|October 12, 2002
Summary
Capillary Isoelectric Focusing (CIEF) offers rapid concentration and separation in a 4-minute process. A novel dialysis interface effectively couples CIEF to Micellar Electrokinetic Chromatography (MEKC) for comprehensive 2D separations.
Area of Science:
- Analytical Chemistry
- Biochemistry
- Separation Science
Background:
- Comprehensive two-dimensional (2D) separations are crucial for complex sample analysis.
- Capillary Isoelectric Focusing (CIEF) is a powerful technique for protein separation based on isoelectric point (pI).
- Coupling CIEF as a second dimension requires efficient interfacing with first-dimension separation methods.
Purpose of the Study:
- To investigate the concentrating properties of CIEF for 2D separation.
- To develop a novel interface for coupling CIEF to a first-dimension separation.
- To demonstrate the application of coupled Micellar Electrokinetic Chromatography (MEKC) and CIEF for protein digest analysis.
Main Methods:
- Utilized Capillary Isoelectric Focusing (CIEF) with continuous whole-column-imaging detection.
- Developed a novel interface using a 10-port valve and two conditioning loops for sample collection and dialysis.
- Coupled Micellar Electrokinetic Chromatography (MEKC) as the first dimension with CIEF as the second dimension.
- Performed dialysis desalting and carrier ampholyte addition within the interface loops.
Main Results:
- Achieved concentration, separation, and detection within 4 minutes in a 300 µm inner diameter capillary.
- Demonstrated that peak broadening during dialysis did not significantly impact CIEF separation due to its concentrating effect.
- Successfully separated protein digests using MEKC followed by whole-column-imaged CIEF.
- Validated the effectiveness of the dialysis interface for general coupling of CIEF to microscale separations.
Conclusions:
- The CIEF system exhibits significant concentrating properties suitable for 2D separation.
- The developed dialysis interface enables seamless coupling of MEKC and CIEF.
- This approach facilitates comprehensive 2D analysis of complex samples like protein digests.
- The interface allows general coupling of whole-column-imaged CIEF to microscale separations.
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