High-Performance Liquid Chromatography Separation and Purification of Cacao (Theobroma cacao L.) Procyanidins According to Degree of Polymerization Using a Diol Stationary Phase

A new chromatographic approach for separating cacao procyanidins according to their degree of polymerization has been developed. It utilizes diol stationary phase columns operating in normal phase mode with a binary gradient of acidified acetonitrile and methanol−water. Performance of the diol stationary phase was evaluated on an analytical scale utilizing classical chromatographic conditions for the normal phase separation of procyanidins according to their degree of polymerization. The new separation approach was developed on an analytical scale but further extended to the preparative scale. These newly developed analytical and preparative high-performance liquid chromatography procedures were successfully applied to the separation, as well as isolation, of cacao procyanidins from unfermented cacao seeds. The degree of polymerization associated with each molecular weight fraction was determined by mass spectrometry. Keywords: Diol; HPLC; HPLC-MS; normal phase; procyanidin; preparative HPLC; flavanols; cocoa