Project Summary/Abstract The overall goal of the proposed project is to develop a new spectroscopy-based technology that will allow for in situ structural determination of hitherto unknown analytes within the liquid chromatography-mass spectrometry (LC-MS) framework. The proposed development is built on the foundation of existing infrared action spectroscopy schemes that provide this capability in a limited manner within specialized research laboratories, and aims to make it broadly useful for metabolomic studies, the discovery of new bioactive molecules from natural sources and other instances where LC-MS is currently used. The specific aims of the project involve developing the methods and instrumentations necessary to obtain a well-resolved IR spectrum of a mass-selected analyte for unambiguous structural identification within the short timeframe dictated by LC-MS. This requires improvements in data acquisition rate of about two orders of magnitude, which will come from faster wavelength tuning rates and enhanced signal-to-noise ratio. To achieve such dramatic improvements, a major component of this project involves transitioning from using a low-repetition-high-pulse-power laser to a high-repetition-lower-pulse-power laser for acquiring the IR spectrum. This will necessitate a complete redesign of the existing experimental setup and approach. Moreover, the proposed developments will involve, to our knowledge, the first instance of applying modulation techniques and lock-in detection to infrared action spectroscopy. If successful, the proposed project will lead to dramatic enhancements in the capability of mass spectrometry, pushing it into the realms of NMR in terms of structural elucidation ability while retaining its high sensitivity, mixture tolerance, and sample throughput rate characteristics. For example, in natural products research the vast majority of the metabolite signals present in the mass spectra of a single sample remain structurally unknown due to the extreme cost such detailed analysis would currently entail. Similarly, in the development of new drugs, new unknown metabolites are generated and require complete characterization. The proposed developments would directly address these needs by providing spectroscopically based chemical structure information within the existing and well-developed LC-MS framework.