Uncovering the origin of impurities in supercritical fluid chromatography coupled to high-resolution mass...

Maintaining low background levels is essential for analytical instruments, such as in supercritical fluid chromatography (SFC) coupled to high-resolution mass spectrometry (HRMS). An approximate tenfold increase of the background was observed in our laboratory when a new CO₂ cylinder with a dip tube was installed on a SFCHRMS system. Most analyte peaks were obscured. This study aimed to find the cause of the elevated background. Accordingly, the entire system was cleaned and each module, including the ion source, back pressure regulator, column, autosampler, solvent pumps, CO₂ pump, and cylinder, were assessed. The troubleshooting suggested that the source of the contamination was the CO₂ cylinder. A statistically significant (p < 0.05) elevation in the background was observed when liquid CO₂ was used as mobile phase. The ions present above normal background levels were found to have m/z of 492.3439, 495.2934, 371.2434, and 490.3381. Annotation of the most abundant ion (m/z of 490.3381) based on m/z, isotope pattern, fragmentation pattern, and collision cross section, suggested structures resembling derivatives of sphingo- and glycolipids. A possible source could be degradation products formed during bioethanol production, the process from which the CO₂ in the cylinder originates. Noteworthily, another SFCHRMS system that uses condensed gaseous CO₂ instead of liquid CO₂, has so far not resulted in any contamination-related issues. To prevent contamination in sensitive SFC-HRMS systems, it is essential to assess the purity of the CO₂ before use, especially if liquid CO₂ from a cylinder with dip tube is used.