Abstract
The difference between normal-phase and reversed-phase high-performance liquid chromatography (HPLC) is explained. This chapter focuses on reversed-phase HPLC. The mobile phase in which the metabolites are dissolved, and the stationary phase with which they interact are described. The particles to which the stationary phase is bound are also described. HPLC is compared with the more modern ultra-high-performance liquid chromatography (UHPLC). The change in the particles that created UHPLC is explained. Variation in the rate of migration, due to interaction with the stationary phase, is explained. The effect of the ratio of polar to nonpolar solvents on migration is discussed. Methods for detection, including mass spectrometry to create liquid chromatography–mass spectrometry, are presented. Interpretation of a chromatogram is explained. An example of UHPLC to assay for chelation of iron by a siderophore is presented. Summaries of the preparation of analytes and the chromatographic parameters are included. Chromatograms that demonstrate chelation of iron are presented.
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