title: Determination and characterization of phytochelatins by liquid chromatography coupled with on line chemical vapour generation and atomic fluorescence spectrometric detection.
abstract: Liquid chromatography (LC) coupled on line with UV/visible diode array detector (DAD) and cold vapour generation atomic fluorescence spectrometry (CVGAFS) has been developed for the speciation, determination and characterization of phytochelatins (PCs). The method is based on a bidimensional approach, e.g. on the analysis of synthetic PC solutions (apo-PCs and Cd(2+)-complexed PCs) (i) by size exclusion chromatography coupled to UV diode array detector (SEC-DAD); (ii) by the derivatization of PC -SH groups in SEC fractions by p-hydroxymercurybenzoate (PHMB) and the indirect detection of PC-PHMB complexes by reversed phase liquid chromatography coupled to atomic fluorescence detector (RPLC-CVGAFS). MALDI-TOF/MS (matrix assisted laser desorption ionization time of flight mass spectrometry) analysis of underivatized synthetic PC samples was performed in order have a qualitative information of their composition. Quantitative analysis of synthetic PC solutions has been performed on the basis of peak area of PC-PHMB complexes of the mercury specific chromatogram and calibration curve of standard solution of glutathione (GSH) complexed to PHMB (GS-PHMB). The limit of quantitation (LOQ) in terms of GS-PHMB complex was 90 nM (CV 5%) with an injection volume of 35 microL, corresponding to 3.2 pmol (0.97 ng) of GSH. The method has been applied to analysis of extracts of cell cultures from Phaeodactylum tricornutum grown in Cd-containing nutrient solutions, analysed by SEC-DAD-CVGAFS and RPLC-DAD-CVGAFS.
authors.affiliation: Italian National Research Council, CNR-Istituto per i Processi Chimico-Fisici, Laboratory of Instrumental Analytical Chemistry, Via G. Moruzzi 1, 56124 Pisa, Italy. emilia@ipcf.cnr.it
authors.forename: Emilia
authors.initials: E
authors.lastname: Bramanti
authors.forename: Daniel
authors.initials: D
authors.lastname: Toncelli
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Morelli
authors.forename: Leonardo
authors.initials: L
authors.lastname: Lampugnani
authors.forename: Roberto
authors.initials: R
authors.lastname: Zamboni
authors.forename: Keith E
authors.initials: KE
authors.lastname: Miller
authors.forename: Joseph
authors.initials: J
authors.lastname: Zemetra
authors.forename: Alessandro
authors.initials: A
authors.lastname: D'Ulivo
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D005978:Glutathione / Q000032:analysis*; D054811:Phytochelatins; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013054:Spectrophotometry, Atomic / Q000379:methods
chemical_list: D054811:Phytochelatins; D005978:Glutathione