Cocoa beans contain secondary metabolites ranging from simple alkaloids to complex polyphenols with most of them believed to possess significant health benefits. The increasing interest in these health effects has prompted the need to develop techniques for their extraction, fractionation, separation, and analysis. This work provides an update on analytical procedures with a focus on establishing a gentle extraction technique. Cocoa beans were finely ground to an average particle size of <100 μm, defatted at 20 °C using n-hexane, and extracted three times with 50 % aqueous acetone at 50 °C. Determination of the total phenolic content was done using the Folin-Ciocalteu assay, the concentration of individual polyphenols was analyzed by electrospray ionization high performance liquid chromatography-mass spectrometry (ESI-HPLC/MS). Fractions of bioactive compounds were separated by combining sequential centrifugal partition chromatography (SCPC) and gel permeation column chromatography using Sephadex LH-20. For SCPC, a two-phase solvent system consisting of ethyl acetate/n-butanol/water (4:1:5, v/v/v) was successfully applied for the separation of theobromine, caffeine, and representatives of the two main phenolic compound classes flavan-3-ols and flavonols. Gel permeation chromatography on Sephadex LH-20 using a stepwise elution sequence with aqueous acetone has been shown for effectively separating individual flavan-3-ols. Separation was obtained for (-)-epicatechin, proanthocyanidin dimer B2, trimer C1, and tetramer cinnamtannin A2. The purity of alkaloids and phenolic compounds was determined by HPLC analysis and their chemical identity was confirmed by mass spectrometry.
Keywords: Cocoa proanthocyanidins; Gel permeation chromatography; Sequential centrifugal partition chromatography; Total phenolic content.