Optimization of sensitivity and separation in capillary zone electrophoresis with indirect fluorescence detection

Abstract

Laser-induced indirect fluorescence detection can be used a general detector in capillary zone electrophoresis. Indirect fluorescence detection, where a fluorophore is used as the principal component of the electrophoretic buffer, allows the visualization of nonfluorescing ions through charge displacement of the fluorophore. Stabilization of laser power improves the dynamic reserve to 10/sup 3/ without the complication of double-beam correction techniques. A modification of the CZE power supply allows the use of dilute solutions and narrow bore capillary tubing (< 15 ..mu..m i.d.). The stability of the background fluorescence is improved by silylation of the fused silica capillary. These modifications improve the detection limits for many anions, nucleotides, and proteins to the range of 50-100 amol of sample injected. Additionally, this improvement in sensitivity results in a concomitant increase in separation efficiency. The electrophoretic separation can be optimized by calculating effective mobilities of ions based on their dissociation as a function of pH. More than 300,000 theoretical plates are observed in a separation of the nucleotide 5'-monophosphates optimized in this manner.