Isolation of Plastoquinone from Spinach by HPLC

We report a method for the purification of plastoquinone-9 (PQ), a prenylquinone cofactor involved in the photosynthetic electron transport chain. The described procedures relies on spinach-chloroplast isolation followed by PQ extraction and chromatographic fractionation. Extraction of PQ was achieved using partition of chloroplast suspension with methanol:petroleum ether. This procedure removed large amounts of green pigments from the extract and thus facilitates the subsequent chromatographic isolation of PQ. To obtain pure PQ, the developed extraction was combined with a two-step chromatographic approach using orthogonal stationary phases, i.e. alumina and octadecylsilane (C18). A small scale protocol for analytical reverse-phase high performance liquid chromatography (RP-HPLC), which may be implemented in most laboratories equipped with conventional systems, is described. The reported methodology represents a valuable tool for the fast production of small amounts of PQ, for which there are no commercial standards available.