Determination of Zinc(ii) Chlorophylls and Their Derivatives by High Performance Liquid Chromatography With Fluorometric Detection

Abstract

Abstract-An HPLC method with fluorometric detection was developed for the determination of zinc (II) chlorophyll a and b ( Zn-chl-a, b ) and zinc(II) chlorophyll ins ( Zn-chins ). The analytical values by the HPLC method with UV and fluorometric detection were compared with those obtained by the spectrophotometric method. The values obtained by the HPLC method using a fluorometric detector were the most reproducible of three analytical methods. It was confirmed that the interferences from the absorption of coexistent pigments are removed by separation using HPLC and the overlapping of peaks from other metallochlorophylls is avoided by using a fluorescence spectromonitor. chlorin e6 ( Zn-chl-e6 ) and zinc(II) rhodin g7 ( Zn-rho-g7 ). The good separation was achieved with C18-bonded silica column and a mobile phase of acetone-methanol for Zn-chls and of methanol-water for Zn-chins. Tailing of peaks was observed for Zn-chlns due to dissosiation of protons in the carboxylic acid of these metallochlorophyllins.The pH value of the mobile phase was adjusted to 3.2 with acetic acid to prevent the release of protons in carboxyl groups. Under these HPLC conditions, Zn-chls and Zn-chins in unknown samples were determined by reversed-phase HPLC with fluorometric detection. EXPERIMENTAL Reagents and apparatusAll chemicals were of analytical-reagent grade and used as supplied without further purification. Organic solvents were purchased from Kanto Chemical Co. Inc. and of LC quality.The semi-preparative HPLC is a liquid chromatograph (Yanaco) equipped with a Develosil ODE column (36 cm x2.4 cm i.d., Nomura Chemical Co. ,Aichi), a VL-611 sample injector (1000mm3 loop, Jasco) and an M-315 (Shodex) variable wavelength detector. The flow rate of the mobile phase for Znchls, acetone-methanol, was 8.0 cm3min-'. The flow rate of the mobile phase for Zn-chins, methanol-H2O containing acetic acid (1% v.), was 3.0 cm3min' . The analytical HPLC instrumentation consisted of a Yanaco L-4000W pump, a Rheodyne 7120 injector with a lOOmm3 injection loop and a Shodex M-315 variable wavelength detector or a Shimadzu RF -530 fluorescence spectromonitor. A prepacked column purchased from GL Sciences was Inertsil ODS-2 which was reversed phase, 5-,u m spherical, octadesil silica in a 250 mm x4.6 mm i.d. column. The column temperature was set at 35 °C by dipping in a thermostat and the flow rate was 1.4 cm3min-' for Zn-chls and 1.0 cm3min' for Zn-chlns. Solvents used as a mobile phase were degassed every time before use by ultrasonically vibrating their containers.Absorption spectra were recorded with a U-2000 spectrophotometer (Hitachi) using 1-cm quartz cells. Preparation of zinc(II) chioro h llsThe preparation of Pheophytin a and b was described in the previous paper [8].Zinc(II) chlorophylls ( Zn-chls ) were prepared by refluxing a mixture of pheophytins dissolved in chloroform and zinc(II) acetate dissolved in methanol at 60 °C for 30 min. All operations were carried out in the dark in a nitrogen atmosphere. The formation and purity of Zn-c...

Fluorescence Quenching Reaction of Chlorophyll a by Tris(acetylacetonate)Iron(III) in Various Solvents

“…Chlorophyll a and its derivatives are not just only frequently used in electron transfer but also being a donor molecule in energy transfer process in plants (Xiaoqing et al 2007). Lots of researches try to replace the metal center of Mg with other transition metal ions in order to explore photo-physical properties of its derivatives (Li & Inoue 1991;Medforth et al 1997;Nonomura et al 1994;Yamashita & Inoue 1991). Nevertheless, only few researches focus on electron transfer of chlorophyll a to Fe (III).…”