Step by Step Operation of ULTIMATE uHPLC Caffeine Analysis Sample preparation: Prepare a solution of caffeine in a suitable solvent. A common solvent system for caffeine analysis is a mixture of water and acetonitrile. Instrument setup: Install the appropriate column and connect the instrument to the computer running the Chomeleon software. Configure the instrument parameters, such as flow rate, column temperature, and detector settings. Method development: Develop a chromatographic method using the Chomeleon software. This involves optimizing the separation conditions, such as the mobile phase composition, column temperature, and flow rate. Sample injection: Inject the caffeine solution into the instrument and start the chromatographic run. Data acquisition: The instrument will acquire the data generated by the detectors, such as UV-Vis and mass spectrometer, during the chromatographic separation. Data analysis: The data can be analyzed using the Chomeleon software, which includes processing, peak detection, integration, and quantification of the separated caffeine peak. Reporting and storage of results: The results of the analysis can be reported and stored in a variety of formats, such as electronic reports or data files. It is important to follow the instrument's user manual and standard operating procedures when operating the ThermoScientific uHPLC instrument and Chomeleon software to ensure the accuracy and reliability of the results. A solvent system of a mixture of water and acetonitrile (e.g., 60:40) is commonly used for caffeine analysis using this instrument. However, the exact composition of the solvent system may need to be optimized depending on the sample type and the separation requirements.