Development of analytical techniques for the analysis of submicron particles and protein aggregates

The goal of the research presented in this dissertation is to develop analytical techniques for the analysis of heterogeneous mixtures of submicron particles and protein aggregates.;In Chapter 2, a simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis (CE) to take advantage of laser-induced fluorescence (LIF) detection. This technique uses laser ablation with a Nd:YAG laser to create gaps of 14.0 +/- 2.2 mum. These structures have been used for reagent addition for postcolumn derivatization with LIF detection and have been tested for the separation of proteins and amino acids.;In Chapter 3, laser-induced backside wet etching (LIBWE) has been adapted to improve the gap construction technique described in Chapter 2. A capillary filled with a solvent or a dye solution was cut by laser ablation. Gap size was reduced up to 56% in comparison to air cut gaps using only 22% of the laser pulse energy used in Chapter 2. The self focusing ability of the solvents tested due to nonlinear refractive index has been shown to play a role in the LIBWE process. Gaps created in Chapters 2 and 3 could be used to label individual protein aggregates and submicron particles.;Separation and detection of individual submicron polystyrene spheres (110-992 nm) using CE with laser light scattering detection at 90o has been demonstrated in Chapter 4. Particles as small as 110 nm in diameter were detected individually using this method, but 57 nm particles could not be detected individually. Detection efficiencies ranging from 38 to 75% were determined for polystyrene spheres of different sizes. The instrument developed in Chapter 4 has been modified to collect scattered light at two different angles (20° and 90°) and fluorescence (90°) simultaneously. The ability of the new system to separate and detect individual 943 nm fluorescent particles was demonstrated. The smallest diameter particle that could be detected at 20° and 90° by scattering was 80 nm. The ability of the system to separate and detect individual rod-shaped biological particles (tobacco mosaic virus) was investigated.