Separation and Determination of Copper(II) Chlorophylls by Reversed-Phase High Performance Liquid Chromatography

Abstract

Copper(II) chlorophyll a and b, i.e. (pheophytinato a)copper(II) and (pheophytinato b)copper(II), were prepared and purified by semi-preparative reversed-phase high performance liquid chromatography (HPLC). The spectroscopic properties of these compounds, which so far have been little studied, are given in detail. Separation of copper(II) chlorophyll a and b is achieved with a C18-bonded silica column and a mobile phase of acetone-acetonitrile (50:50, v/v). A rapid and accurate HPLC method with visible detection at 425nm has been proposed for the simultaneous determination of copper(II) chlorophyll a and b. Both calibration graphs for copper(II) chlorophyll a and b are linear in the concentration range 0-10μg cm-3. The detection limits of copper(II) chlorophyll a and b are 4.4ng cm-3 within 3.1% relative standard deviation (RSD) (n=10) and 2.3ng cm-3) within 3.3% RSD (n=10), respectively. The HPLC method proposed here has also been evaluated in comparison to the spectrophotometric method. This reversed-phase HPLC method can be performed within a short time period, thus facilitating an accurate routine assay of many samples.