The present work is directed towards preparative isolation of the above impurities and their characterization by spectroscopic techniques like UV, NMR and mass spectrometry. After the collection of each fraction, the solvent is evaporated under vacuum and the purity checked by analytical HPLC. All the collected impurities were found to be over 98% pure. Later the fractions were analyzed by 1H and 13C NMR spectrometry. The spectral data has been used for characterization of the impurities collected by preparative HPLC. The ESI–MS data of the fractions has provided evidence of molecular masses. The 1H NMR data of these impurities has been compared with the values reported in the literature and found to be in good agreement with the minor impurities of astaxanthin[23] and [24].
Mass spectra were recorded using a quadrupole mass spectrometer (QuatroLC, Micromass, UK) equipped with an electro spray ion source. The mass spectra were acquired in positive ion mode over the mass range 50–1000 Da.