Project Summary. Nuclear magnetic resonance (NMR) is among the most powerful analytical techniques ever invented, as recognized by 6 Nobel Prizes for methods development alone. Nonetheless, NMR is notoriously plagued by poor sensitivity. State-of-the-art NMR spectrometers feature detection thresholds of ~1 nanomole for µL sample volumes (~100 nanograms). This places NMR sensitivity many orders of magnitude behind other analytical chemistry techniques such as mass spectrometry, Raman spectroscopy, and fluorescence labeling. Improvements in NMR often focus on using larger magnets, but progress has plateaued; over the last 25 years, the fundamental signal strength has only increased ~2-fold. We seek to fundamentally change the NMR hardware by using diamond films doped with Nitrogen-Vacancy centers to detect nuclear magnetization non-inductively via pulsed optically detected magnetic resonance methods. The form factor of our NMR detector is easily integrated with hyphenation techniques so that samples can be separated into sub-components before analysis. Recently, we built a tabletop microfluidic diamond NMR apparatus with 40 pL detection volume and used it in proof-of-principle analytical chemistry applications including the first 2D NMR spectra acquired by a diamond NMR sensor. In Phase I, we will optimize sensor spectral resolution and sensitivity and validate its operation using metabolite mixtures. This work will place us in the position to deliver our devices to end-users in industry (Merck) and academia (UW) and incorporate feedback to scale up to market. If successful, our prototype could have a profound impact on analytic biochemistry research, by combining mass-spectrometry-level sensitivity with NMR-level accuracy. Specifically, we improve upon existing analytical methods by offering: 1. Greater performance. We offer 1000-fold better sensitivity (pmol instead of nmol) than current NMR spectrometers. This sensitivity approaches that of mass spectrometry but retains benefits of NMR such as non-destructive, absolute quantitation and structural identification. 2. Compatibility with hyphenated separation techniques. Our spectrometer is compact and easily integrated into microfluidic chips for online chromatography-based assays (HPLC) for sample-limited analyses (metabolomics, pharmacodynamics, natural products). 3. Lower cost. The small sample volume in our spectrometer leads to reduced engineering costs, leading to greater affordability compared to current NMR spectrometers.