This report describes laboratory development and process plant applications of Raman spectroscopy for detection of hydrogen isotopes in the Tritium Facilities at the Savannah River Site (SRS), a U.S. Department of Energy complex. Raman spectroscopy provides a lower-cost, in situ alternative to mass spectrometry techniques currently employed at SRS. Using conventional Raman and fiber optics, we have measured, in the production facility glove boxes, process mixtures of protium and deuterium at various compositions and total pressures ranging from 1000 - 4000 torr, with detection limits ranging from 1-2% for as low as 3-second integration times. We are currently investigating fabrication techniques for SERS surfaces in order to measure trace (0.01-0.1%) amounts of one isotope in the presence of the other. These efforts have concentrated on surfaces containing palladium, which promotes hydrogen dissociation and forms metal hydride bonds, essentially providing a chemical enhancement mechanism.
We are currently developing miniaturized, chip-based electrophoresis devices fabricated in plastics for the high speed separation of oligonucleotides. One of the principal advantages associated with these devices is their small sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for oligonucleotides, are done off-chip on a microliter-scale. Our work has focused on the development of capillary nano-reactors coupled to micro-separation platforms, such as micro-electrophoresis chips, for the preparation of sequencing ladders and also, PCR reactions. These nano-reactors consist of fused silica capillary tubes (length equals 10 - 20 cm; id equals 20 - 50 micrometer) with fluid pumping accomplished using the electro-osmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cycle sequencing or PCR amplification of the DNAs. The reactors were interfaced to the micro-electrophoresis chips via capillary connectors micromachined in polymethylmethacrylate (PMMA) using deep X- ray etching (width equals 50 micrometer; depth equals 50 micrometer) and were situated directly on the PMMA-based microchip. This chip also contained an injector, separation channel (length equals 6 cm; width equals 30 micrometers; depth equals 50 micrometers) and a dual fiber optic, near- infrared fluorescence detector. The sequencing nano-reactor used surface immobilized templates attached to the wall via a biotin:streptavidin:biotin linkage produced by PCR using a biotinylated forward primer. Sequencing tracks could be directly injected into gel-filled capillary tubes with minimal degradation in the efficiency of the separation process. The nano-reactor could also be configured to perform PCR reactions by filling the capillary tube with the PCR reagents and template. After thermal cycling, the PCR cocktail could be injected into a capillary tube or a micro-chip device for fractionation. In all cases, the detection of the oligonucleotides was accomplished using ultra-sensitive near- IR fluorescence detection.