Nine Orders of Magnitude Dynamic Range: Picomolar to Millimolar Concentration Measurement in Capillary Electrophoresis with Laser Induced Fluorescence Detection Employing Cascaded Avalanche Photodiode Photon Counters

Abstract

The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. Appropriate scaling of the signals from the five photodiodes produces a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM to the upper limit of 1 mM. Mass detection limits are 120 yoctomoles (70 molecules) injected into the instrument. The very-wide dynamic range instrument was used to study the metabolic products of the fluorescently labeled glycosphingolipid tetramethylrhodamine labeled GM1 (GM1-TMR) produced by single cells isolated from the rat cerebellum.