Preparation and determination of manganese(III) chlorophylls by reversed-phase high performance liquid chromatography

Abstract

Manganese chlorophylls have been synthesized by refluxing a mixture of pheophytins dissolved in acetone and manganese(II) acetate anhydride dissolved in glacial acetic acid. A good separation of manganese(III) chlorophylls has been attained by RP-HPLC using chemically bonded C18 silica as a stationary phase and methanol with 3% acetic acid as a mobile phase. An accurate and rapid HPLC method is described for the simultaneous determination of manganese(III) chlorophyll-a [Mn(III)-chl-a] and manganese(III) chlorophyll-b [Mn(III)-chl-b]. Tailing arising from dissociation of acetate ions is improved by addition of sodium acetate (5×10−3 M) to the mobile phase (acetone: methanol=90∶10, vol/vol). The analytical values obtained by the HPLC method are very close to the calculated contents in all samples, but those obtained by spectrophotometry are high because of the interferences from overlapping of absorption bands. In the proposed HPLC method the calibration graphs of Mn(III)-chl-a and Mn(III)-chl-b are linear in the concentration range 0–20 μg cm−3 with relative standard deviations (n=10) of 3.46% and 4.54%, respectively.