Binary Solvent Effects in Capillary Zone Electrophoresis with Ultrasensitive Near-IR Fluorescence Detection of Related Tricarbocyanine Dyes and Dye-Labeled Amino Acids

Abstract

We have investigated the effects of mixed organic/aqueous carrier buffers on the chromatographic efficiency and detectabilipy of native near-infrared (near-IR) dyes and near-IR dye-labeled amino acids separated using capillary electrophoresis (CE). The near-IR fluorescence detector for this application incorporated a solid-state Ti/sapphire laser for excitation and a single-photon avalanche diode photodetector. The on-column mass detection limits for a series of cationic tricarbocyanine near-IR dyes varied from 350 to 5500 ymol in running buffers comprised of 95/5 methanol/water (pH=9.1). In predominately aqueous running buffers, the detection limits degraded with significant losses in the separation efficiencies. Two near-IR dyes with similar structures but different charges (cationic and anionic) were used to investigate efficiency losses with binary methanol/water running buffers. The results indicated that solute/wall interactions were a major contribution to zone broadening for the cationic dye in high water compositions, resulting in significant losses in the numbers of theoretical plates, while the anionic dye did not exhibit these types of interactions. In order to demonstrate the usefulness of near-IR fluorescence in CE applications, a series of amino acids were labeled with a near-IR fluorescent dye containing an isothiocyanate fuctional group and separated using CE with mixed methanol/water buffers. The fluorescence signal strength and the chromatographic resolution were found to be improved in high methanol compositions in the running buffer