Abstract

Biological samples remain challenging in proteomic separations due to their complexity and large concentration dynamic range. Improvements to separation power are needed to interrogate proteomes more deeply and facilitate the advancement of biomarker discovery for personalized medicine.

Current online multidimensional separations require compromise; long analysis times if the second dimension (2nd-D) must be regenerated between injections, or reduced separation efficiency if the 2nd-D is operated rapidly. Using an array of capillaries as the 2nd-D, operated in parallel, allows fast sampling of the first dimension (1st-D). This relaxes the constraints on the 2nd-D separation, allowing it to operate at optimal separation conditions that would otherwise be sacrificed for speed. This configuration allows total separation times to approximately equal to the 1st-D separation time.

We have developed a novel interface that enables continuous sampling of a 1st-D separation by a 2nd-D capillary array for rapid, high peak capacity two dimensional (2D) separations, based upon automated precision positioning of capillaries. Within a laminar flow regime, a capillary electrophoresis (CE) 1st-D separation was coupled to an array of eight independent CE 2nd-D separations. The instrument terminus provides laser induced fluorescence detection via a sheath flow cuvette.

Effluent transfer efficiency, from the 1st-D to the 2nd-D, and detection was optimized using visible and fluorescent dye tracers. To that end, this dissertation will discuss characterization of interface and detector parameters, including: inter capillary transverse alignment accuracy, injection distance, injection time, hydrodynamic flow rate, density considerations, inter and intra capillary differences, signal crosstalk and laser intensity.

Separation performance will further be demonstrated using model protein and serum digestates. Each dimension of the 2D instrument will be operated as a one dimensional (1D) instrument to compare against an optimized commercial 1D CE instrument. These results will be used to evaluate the quality of the separations operated in on-line 2D capillary electrophoresis-to-capillary array electrophoresis (CE×CAE) mode. A novel application of the CE×CAE design will be discussed in the spirit of resolving the long standing challenge of migration time reproducibility in CE separations.