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Design and applications of improved hyphenation and separation strategies for multidimensional biomolecule characterization

University of British Columbia

A novel interface for capillary electrophoresis – electrospray ionization – mass spectrometry (CE-ESI-MS) has been developed with the goal of providing a robust and easy-to-use hyphenation strategy that also provides high sensitivity. The interface uses a tapered stainless steel needle that surrounds the capillary terminus, so that the needle interior acts as the CE outlet vial and the outside tip is the electrospray emitter. A chemical modifier solution, introduced via a tee junction, serves to improve the compatibility of the background electrolyte with ESI and allows the interface to operate under conditions where the bulk flow in the capillary is suppressed or reversed. A major novel feature of the interface is the use of a beveled conductive needle as the ESI emitter. Because electric field is highest at the sharpest edge of an electrode, liquid exiting the needle is drawn to the sharpest point of the bevel, whereas for a conventional symmetrically tapered emitter the field is equally distributed around the circumference of the tip. Electrospray performance as a function of flow rate was investigated for a variety of emitter geometries. The beveled emitters provided a more stable and efficient electrospray over a wider range of flow rates than traditional emitters. The second part of the thesis describes the application of the CE-ESI-MS interface with the beveled emitter to a variety of biomolecular species, including peptides, proteins and carbohydrates. These applications include the use of a variety of capillary coatings and separation conditions where the electroosmotic flow is suppressed or even reversed, so that the bulk flow in the CE capillary is away from the interface. The interface was also used to interface capillary isoelectric focusing (cIEF) with MS detection, enabling free solution and fully automated protein analysis that is analogous to 2D gel electrophoresis. The final part of the thesis examines the viability of a two-marker correction strategy for improving retention time reproducibility in reversed-phase liquid chromatography. Although the strategy provided up to a ten-fold improvement in reproducibility for repeated separations, the correction was not successful in relating retention times obtained under different gradient conditions.

Text

2012-02-29

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/31773

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/