Preconcentration techniques in capillary electrophoresis with laser-induced fluorescence detection.

摘要

Capillary electrophoresis (CE) is a separation technique based on the differential migration of solutes in an electric field. In CE, one of the long-standing problems is the poor detection sensitivity, especially when ultraviolet detection is used. Several pre-concentration techniques were utilized in this thesis for enhancing the detection sensitivity of CE with laser-induced fluorescence detection (LIF).; High-salt stacking, originally proposed by Lander et al. , was applied to enrich fluorescently labelled alkylphosphonic acids in micellar electrokinetic chromatography (MEKC). By adding sodium chloride to the sample solution, a discontinuous buffer system was created in the capillary. The difference in conductivity at the sample zone - MEKC buffer zone interface resulted in the deceleration and accumulation of sodium cholate micelles at the zone boundary and the subsequent concentration of hydrophobic analytes. An enrichment of approximately 10 fold was obtained.; Enrichment of analytes can also be achieved based on solid-phase extraction (SPE) in either an off-line or online manner. A 200-mul micropipette tip packed with anion-exchanger beads was used for offline SPE extraction of glyphosate, a herbicide, from a spiked river water sample. Subsequent elution of the retained glyphosate and fluorescent labeling allowed detection of glyphosate in the sub-nanomolar range. An approximate 88-fold preconcentration was observed by using such an off-line resin-packed SPE pipette tip.; SPE-based on-capillary preconcentration was developed by entrapping HPLC bulk packing (PRP-1) into a sol-gel silica monolith in a short segment of capillary and attaching the prepared SPE capillary to the tip of separation capillary. After fluorescent labeling, two herbicides, ampropylfos and glufosinate, were extracted onto the PRP-1 beads, followed by elution and CE-LIF analysis. The charge states of analytes were found to affect the extraction ratio, which can be improved significantly either by reducing the pH or by adding salt to the sample solution. Examination of the PRP-1 packed silica monolith by scanning electron microscopy (SEM) found that PRP-1 beads were encapsulated by a silica shell. A very small fraction of PRP-1 beads had exposed surface for extracting hydrophobic analytes. This problem was solved by partial digestion of the silica shell using a pH 9.4 borate buffer. With significantly increased PRP-1 bead surface and salt effect during sample loading, 1260- and 580-fold enhancement of detection sensitivity were achieved for ampropylfos and glufosinate, respectively.; Finally, in an alternate project, a capillary gel electrophoresis (CGE)-LIF method using a polyethylene oxide (PEO) polymer sieving matrix was developed for separation of five model proteins. For the first time, detergent differential fractionation (DDF) technique was combined with CGE for the sequential fractionation and protein profiling of HT29 human colon adenocarcinoma cell extract.