Miniature In-line Microfluidic LIF Detector

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High-pressure liquid chromatography (HPLC) and subsequent analysis by mass spectrometry (MS) are important, gold-standard techniques in the field of proteomics. This approach has been further enhanced by the introduction of microflow HPLC coupled directly to nanospray MS inlets. Although this can greatly save on sample materials, many peaks that are resolvable with MS are below the current limit of detection for standard or micro UV HPLC detectors. The use of fluorescence detection, which has substantially greater sensitivity, allows on-line, continuous monitoring of the HPLC effluent prior to MS analysis in these micro and nanoflow systems. The coupling of fluorescence detection with MS analysis then allows for multi-dimensional analysis of the sample under investigation: with internal standards, each resolved peak can be quantified prior to MS characterization. To accomplish this advance in MS technology, a miniature in-line microfluidic laser induced fluorescence (LIF) detector was designed and constructed. The key feature of this detector is its portability, which permits easy insertion into the capillary microfluidic flow path after HPLC separation and immediately prior to MS analysis. This is enabled by the use of fiber-optics for both the excitation and emission paths of the laser induced fluorescence detector that allows bulky light sources, optical filtration elements, and photon detectors to be housed remotely from the point of detection. The small (4x4x1cm) prototype detector head consists of an embedded channel to accommodate a short length of silica capillary, with a 385-micron outside diameter and a square 100-micron diameter internal flow path. Perpendicular to the capillary is a second embedded channel to accommodate a 200-micron fused silica optical fiber that is positioned in close proximity to an observation window formed in the capillary by removal of approximately 2 mm of the polyimide coating. This fiber conducts excitation light (405 nm in this instance) to induce fluorescence of the sample analytes that are tagged with naphthalene dicarboxaldehyde (NDA). A second optical fiber, 600-micron fused silica, is also positioned at the same observation point and is perpendicular to both the capillary and the excitation fiber. The light collected by this optical fiber is passed through a thin-film filter to eliminate scattered laser light, and the remaining fluorescent signal is detected using a photomultiplier tube. The signal from the photomultiplier tube can be read by any voltage recorder, digital or analog. The complete detector head, including capillary tubing, interconnects and a light-tight housing is approximately 10 cm in length and 5 cm in cross section. The observation sample volume is approximately 6 nl and a sensitivity of 50 fmol or less was achieved by LIF with NDA peptide labelling of a tryptic digest of apomyoglobin that was used as a model sample for proteomic analysis. Chromatograms show excellent resolution at flow rates of 5 microliters/min. The sensitivity and resolution achieved place this detector at the current state-of the-art, which together with its compactness and portability make it a significant advance in HPLC/MS technology. Future efforts will include incorporating a second wavelength for excitation and detection, in particular the detection of native tryptophan fluorescence.