Liz Knisley HPLC and LC-MS Introduction Liquid chromatography is a separation technique that relies on the use of a liquid mobile phase and a solid/liquid stationary phase. This type of chromatography is similar to gas chromatography but has a different type of mobile phase and column designs. A key aspect of chromatography is the detector used at the end of separation. Detectors range from mass analyzers to fluorometers and are used to identify separated analytes using different techniques. High performance liquid chromatography (HPLC) is a technique that uses high pressure to quickly separate analytes in a mixture. In this lab we used HPLC and liquid chromatography mass spectroscopy (LCMS) to separate and analyze caffeine standards. Procedure LCMS Turn on Nitrogen Open software and activate profile Build acquisition set to program automated sampling Place samples into auto sampling vial positions Run samples using computer program Analyze data using list data, peak list, and data file Deactivate LCMS and shut down HPLC Degas methanol and water if there is not enough Turn on computer Check pump lines A and B to ensure no gas bubbles o Execute purge with pump if gas bubbles exist o Purge lines by opening valve and hitting purge button o Close valve and quickly hit purge button again Turn pump back on Turn detector on Select method and align parameters Run standards o 50ppm, 40ppm, 30ppm, 20ppm, and 10ppm Run samples from regular tea and decaf tea Data All data for HPLC can be found in my lab notebook under the section “Lab #2”. The LCMS was down during our lab session and was not back up for most of the semester. Conclusion Due to the LCMS being down for most of the semester, including during my lab partner and I’s lab session assigned for the instrument caused us to not get a chance to experience the LCMS. However, the HPLC was fairly easy to use and did not give us many issues due to no air bubbles in the lines and everything was functioning properly. Because of this instrument being so user friendly and fast at getting results, we had chosen to use this instrument for our real world project.