Improved Laser-Induced Fluorescence Detection for Capillary Electrophoresis

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The introduction of laser-induced fluorescence (LIF) detection to separation science has greatly improved detection sensitivity for a variety of analytes. The range and application of this type of detector has been further enhanced by the development of fluorophore labels with absorption spectra matched to known laser lines. Although a number of commercial LIF detectors are available, laboratory-designed, purpose-built systems can offer improved sensitivity as well as added function. In recent years, researchers in our group developed an ultrasensitive LIF capillary detector for applications in the separation and biological sciences that achieved a sensitivity of 450 fg/ml of Substance P. To address the need for internal standards, our group introduced a second wavelength capability into the design for simultaneous measurement of internal standards and unknown analytes within the same sample. The internal standards were labeled with Bimane while human plasma samples were labeled with AlexaFluor633. Each sample was spiked with a mixture containing 100 pg of each standard and injected into a capillary electrophoresis system. The samples were run at 75 mA constant current and the resolved peaks detected on-line with a flow-cell set 60 cm from the inlet. The fluorescent signals were measured by the two-color detector, consisting of a 408-nm diode pumped solid state laser and a 633-nm helium-neon laser co-linearly combined and brought to common focus at the flow-cell. Emitted light was collected with an optical fiber, in close proximity to the flow-cell, and positioned orthogonally to both the flow cell and the laser excitation beams. The beam exiting the fiber was collimated and passed through a 417-nm long pass Raman edge filter combined with a 633-nm laser notch filter. The resulting signal was transmitted via a second optical fiber to the entrance slit of a CCD spectrometer equipped with a data acquisition board and a Labview interface. The two resulting chromatograms were plotted as fluorescence units versus time with stacked traces. This new laser induced fluorescence (LIF) detector improves on our previous work by allowing the simultaneous detection of internal standards that are labeled with one fluorophore (Bimane) and serum samples labeled with a different fluorophore (AlexaFluor633). This enables investigators to perform multi-analyte analyses on a variety of biological specimens. Quantification of the natural materials is determined by calculating peak areas and directly comparing them with those obtained with the standards. The advantage of the two-color detector is that direct calculation of unknowns can shorten analytical time and negate the need for additional standard or calibration runs. An added advantage of two-color LIF detection is that a high degree of sensitivity can be achieved during detection and that several standards can be introduced, thus allowing for multi-analyte identification and quantification. Additionally, the simultaneous detection of standards and unknowns, within the same sample, greatly reduces analytical time. Finally, the reduced analytical time plus the in-built quality control makes this approach ideal for clinical studies and patient monitoring. A new version of the two-color detector optimized for detection from a square capillary of 50 micron internal diameter has now been designed, assembled, and characterized. The laser wavelengths have also been changed to 660 nm and 780 nm to reduce the contribution from sample autofluorescence. After investigating a number of different configurations, we have chosen a system which uses high numerical aperture collimating lenses, together with a pinhole to exclude unwanted background. The collected light is directed onto photomultiplier tubes, one for each wavelength, in contrast to the CCD detector of the earlier design. These changes, together with the incorporation of newly available solid-state lasers, have resulted in a substantial decrease in the overall size of the detector, and hence the distance from the separation to the detection in the on-line system. In this configuration, the detector is optimized for use with a nanoflow HPLC system; incorporation of the detector into this system should follow shortly.