BINARY SOLVENT EFFECTS IN CAPILLARY ZONE ELECTROPHORESIS WITH ULTRASENSITIVE NEAR-IR FLUORESCENCE DETECTION OF RELATED TRICARBOCYANINE DYESAND DYE-LABELED AMINO-ACIDS

We have investigated the effects of mixed organic/aqueous carrier buff ers on the chromatographic efficiency and detectability of native near -infrared (near-IR) dyes and near-IR dye-labeled amino acids separated using capillary electrophoresis (CE). The near-IR fluorescence detect or for this application incorporated a solid-state Ti/sapphire laser f or excitation and a single-photon avalanche diode photodetector. The o n-column mass detection limits for a series of cationic tricarbocyanin e near-IR dyes varied from 350 to 5500 ymol in running buffers compris ed of 95/5 methanol/water (pH = 9.1). In predominately aqueous running buffers, the defection 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 eff iciency 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, resul ting 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 applicati ons, a series of amino acids were labeled with a near-IR fluorescent d ye containing an isothiocyanate functional group and separated using C E with mixed methanol/water buffers. The fluorescence signal strength and the chromatographic resolution were found to be improved in high m ethanol compositions in the running buffer.