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
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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
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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.