HPLC Rotamer Separation and Determination of an Extract Fraction of a Microbial Pigment

A bioactive red pigment fraction was extracted from a marine bacterium. The preliminary experimental data suggested that the extracted fraction may belong to a member of prodigiosin family. An HPLC rotamer separation method was developed to define the chemical identity of the isolated bacterial material. The chromatogram of the pigment showed a peak absorption at 530 nm and a typical retention time 5.6 min, same as the prodigiosin standard, when analyzed with a C-18 column and an isocratic mobile phase of acetonitrile:water (80:20, v/v, 1ml/min, pH=7). Upon lowering the pH value of the mobile phase to 3 with HCl, the HPLC chromatogram of the nominally purified pigment showed two distinct absorption peaks. The separation of the parent (α rotamer) and daughter (β rotamer) peaks was pH- and temperature-dependent. At the optimal condition (5°C and pH=3), the size of the β peak is approximately 30% of the α peak. All these parameters of rotamer separation of the unknown microbial pigment are identical to that ...