title: High-performance liquid chromatography method for ferric iron chelators using a post-column reaction with Calcein Blue.
abstract: Iron (Fe) is an essential element for higher plants, which take it up from the soil at the root surface and transport it to shoots through the xylem. Fe(III) chelators, such as organic acids and phytosiderophores, play important roles in the acquisition and transportation of Fe(III). Therefore, a selective and sensitive method for analyzing Fe(III) chelators is required to study the many Fe-related physiological mechanisms in plants. A novel analytical approach employing a high-performance liquid chromatography post-column method with fluorescence detection was developed to separate and detect Fe(III) chelators. This method takes advantage of the quenching of the fluorescence of Calcein Blue (CB) that occurs with the formation of an Fe(III)-CB complex and the dequenching that occurs with the release of CB as a result of competition for Fe(III) between CB and an Fe(III) chelator. This simple experimental method does not require complicated pretreatments and can selectively detect Fe(III) chelators according to their Fe(III)-chelating ability. The detection limit for citric acid using this method was 72pmol. Furthermore, this method can also detect unknown Fe(III) chelators that exhibit a high affinity for Fe(III). The method was evaluated with xylem sap of barley, which was shown to contain several Fe(III) chelators.
authors: Department of Applied Biological Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan.
authors: Tomoko
authors: T
authors: Ariga
authors: Department of Applied Biological Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan.
authors: Kyoko
authors: K
authors: Ito
authors: Department of Applied Biological Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan.
authors: Yuki
authors: Y
authors: Imura
authors: Department of Applied Biological Chemistry, The University of Tokyo, Yayoi 1-1-1, Bunkyo, Tokyo 113-8657, Japan. Electronic address: ayoshim@mail.ecc.u-tokyo.ac.jp.
authors: Etsuro
authors: E
authors: Yoshimura
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005452:Fluoresceins / Q000032:analysis* / Q000378:metabolism; D001467:Hordeum / Q000737:chemistry / Q000378:metabolism; D007502:Iron Chelating Agents / Q000032:analysis* / Q000378:metabolism; D057230:Limit of Detection; D015203:Reproducibility of Results; D052584:Xylem / Q000737:chemistry
chemical_list: D005452:Fluoresceins; D007502:Iron Chelating Agents
keywords: Amino acids; Carboxylic acids; Fluorescence quenching; HPLC; Organic acids; Phytosiderophores