Stepwise pH-gradient elution for the preparative separation of natural amthraquinones by multiple liquid-liquid partition

Preparative-scale separation of substituted anthraquinones by multiple liqu id-liquid. partition was studied using isopropylmethyl ketone (IMK)/aqueous phosphate buffer (aq.) as the solvent system and the Hietala apparatus wit h 100 partition units as the partition equipment. The lower (aq.) phase was chosen as mobile, while the upper (IMK) phase remained stationary. Hence, the principle of stepwise pH-gradient elution could be utilized to separate the components in two complex mixtures of hydroxyanthraquinones and hydrox yanthraquinone carboxylic acids, isolated from the fungus Dermocybe sanguin ea. In spite of the nonlinearity of the partition isotherms for these anthr aquinones, implying deviations from the Nernst partition law, remarkable se parations were achieved for the components in each mixture. Every anthraqui none carboxylic acid showed markedly irregular partition behavior, appearin g in the effluent as two more or less resolved concentration zones. Such sp litting was attributed to the formation of relatively stable sandwich-dimer s, which were in a slow equilibrium with the monomers in the more nonpolar organic phase. At lower pH-values, only the polar monomers were distributed into the mobile aqueous phase and moved forward, whereas the nonpolar sand wich-dimers remained almost entirely in the stationary organic phase and la gged behind. When the pH of the mobile aqueous phase was raised high enough , the firmly linked dimers were monomerized and emerged from the apparatus as a second concentration profile. Intermolecular hydrogen bonding and pi-p i interaction between the pi -systems of two anthraquinone molecules in a p arallel orientation were considered responsible for the nonlinear and marke dly irregular partition behavior of the natural anthraquinones studied. The nonlinearity of the partition behavior of the hydroxyanthraquinones lackin g the carboxyl group, appeared merely as excessive broadening of the experi mental concentration profile, which impaired the resolution between the com ponents only insignificantly. Thus, e.g. the main components, dermocybin an d emodin, could both be obtained from Separation 1 in a purity of at least 99%.