Capillary ultrahigh performance liquid chromatography with elevated temperature for sub-one minute separations of basal serotonin in submicroliter brain microdialysate samples.

Improving the time resolution in microdialysis coupled to high performance liquid chromatography (HPLC) requires that the volume of the separation system be decreased. A low-volume separation permits smaller microdialysate volumes to be injected without suffering a sensitivity loss from dilution. Thus, improved time resolution can be achieved with offline analysis simply by decreasing the separations system volume. For online (near real-time) analysis, there is a further requirement. The separation speed must be at least as fast as the sampling time. Here, the combined use of high column pressures and temperatures, sub-2-µm stationary phase particles, capillary columns, and sensitive, low dead-volume detection resulted in a retention time for the neurotransmitter serotonin of less than 1 min in a 500 nL dialysate sample volume. Two sensitive detectors, photoluminescence following electron transfer (PFET) and electrochemical, were used for the detection of subnanomolar concentrations of serotonin in brain microdialysate samples. The general principles developed are applicable to a wide range of separations with the additional advantages of increases in sample throughput and decreases in mobile phase usage.

Carbon Fiber/Epoxy Composite Ring-disk Electrode: Fabrication, Characterization and Application to Electrochemical Detection in Capillary High Performance Liquid Chromatography.

Carbon fiber/epoxy composite materials, which are manufactured using the pultrusion process, are commercially available in various shapes and sizes at very low cost. Here we demonstrate the application of such a material as an electrochemical detector in a flow system. Cyclic voltammetry shows that the material's electrochemical behavior resembles that of glassy carbon. Using tube and rod composites, we successfully fabricated a ring-disk electrode with a 20 µm gap between the ring and the disk. The narrow gap is favorable for mass transfer in the generator-collector experiment. This composite ring-disk electrode is assembled in a thin-layer radial-flow cell and used as an electrochemical detector. The disk electrode, placed directly opposite to the flow inlet, is operated as a generator electrode with the ring electrode being a collector. The high collection efficiency on the ring electrode (0.8 for a chemically reversible species) enhances the detection selectivity.

Electrochemical and optical detectors for capillary and chip separations.

In separations in capillaries or on chips, the most predominant detectors outside of the field of proteomics are electrochemical (EC) and optical. These detectors operate in the µM to pM range on nL peak volumes with ms time resolution. The driving forces for improvement are different for the two classes of detectors.With EC detectors, there are two limitations that the field is trying to overcome. One is the ever-present surface of the electrode which, while often advantageous for its catalytic or adsorptive properties, is also frequently responsible for changes in sensitivity over time. The other is the decoupling of the electrical systems that operate electrokinetic separations from the system operating the detector.With optical detectors, there are similarly a small number of important limitations. One is the need to bring the portability (size, weight and power requirements) of the detection system into the range of EC detectors. The other is broadening and simplifying the applications of fluorescence detection, as it almost always involves derivatization.Limitations aside, the ability to make detector electrodes and focused laser beams of the order of 1 µm in size, and the rapid time response of both detectors has vaulted capillary and chip separations to the forefront of small sample, fast, low mass-detection limit analysis.