4 mS/cm). Besides, IEX shows poor scalability and is the slowest method

investigated (see Section 2). The use of NPC and ATPS in the purification of betanin produced the worst results. The PEG/(NH4)2SO4 system is based on the differential partitioning of betalains and sugars between the aqueous phases (Chethana et al., 2007 and Neelwarne and Thimmaraju, 2009). Consequently, the presence of large amounts of dextrin in sample C compromises the partitioning, making this approach inefficient. Results reported here are yet to be compared to those obtained using countercurrent chromatography, which has been applied effectively for the purification of betanin and its epimer on the preparative scale (Degenhardt and Winterhalter, 2001, Schliemann and Strack, 1998 and Wybraniec et al., 2010). Betalain

optical isomers can be resolved by several chromatographic methods in the absence of Ibrutinib price chiral stationary phases (Schliemann et al., 1999 and Wybraniec et al., 2009). For example, betanin has a shorter retention time than isobetanin (ΔtR = 0.3 min) Olaparib in the RP-HPLC analysis reported in this work. This difference has been rationalised in terms of a stronger interaction of iBn with the non-polar stationary phase, but no quantitative data is given whatsoever ( Schwartz and Von Elbe, 1980 and Wybraniec, 2005). Therefore, we examined this effect quantitatively through theoretical calculations. Due to the size of betanin, geometry ID-8 optimisation and frequency

calculations were conducted using the semi-empirical method PM6 (Stewart, 2007); however, the energies and the physicochemical properties in water were determined at the SMD/M06-2X/6-311++G(d,p) level (Marenich et al., 2009, Zhao and Truhlar, 2008a and Zhao and Truhlar, 2008b). Furthermore, we limited ourselves to two conformational isomers of both Bn and iBn, namely the axial carboxyl and equatorial carboxyl conformers (Scheme S1, coordinates of optimised geometries are given in the Supplementary data). Due to the lack of structural parameters for betanin and isobetanin, the structure optimisation was verified by comparison of the theoretical electronic transition wavelengths (λTh), determined using the SMD/ZIndo/S approach ( Marenich et al., 2009 and Zerner, 1991), with experimental absorption maxima. The equatorial carboxyl conformers of both Bn and iBn are more planar than the corresponding axial isomers. Results obtained for the equatorial carboxyl conformers of both Bn and iBn are in excellent agreement with experimental data (Table 3). This correspondence is obtained exclusively when fully protonated forms of Bn and iBn are used (pK  a of the carboxylic groups of betanin is 3.4) ( Nilsson, 1970). The absorption maximum is not pH-dependent in the 3–7 pH range ( Azeredo, 2009); however, the results can be rationalised based on the occurrence of hydrogen bonds with strong ion-dipole character between the carboxylate groups and water.