Column chromatography and verification of phytosiderophores by phenylisothiocyanate derivatization and UV detection

A high-performance liquid chromatographic method was developed for the separation of phytosiderophores and for verification of the Fe3+-complexing ability of chromatographic separates. The chromatographic procedure involves separation on a C-18 reversed-phase column by linear gradient elution with sodium acetate (pH 6.4) and methanol with UV detection, following pre-column derivatization of the phytosiderophores with phenylisothiocyanate (PITC). A modification of the procedure involving pretreatment of the sample with Fe3+, which blocks the derivatization by PITC, was used to confirm whether a given chromatographic peak was attributable to an Fe3+-complexing compound. The practical detection limit was approximately 100 pmol of phytosiderophore. (C) 1999 Elsevier Science B.V. All rights reserved.