Characterization of carotenes in a combination of a C18HPLC column with isocratic elution and absorption spectra with a photodiode-array detector

Carotenes have attracted much attention in recent years for their biological function in processes such as photosynthesis. The characterization of carotenes is difficult, however, because they consist of only carbon and hydrogen atoms, without oxygen. In the present study, we systematically examined the chemical structures of more than 30 carotenes, including most of the carotenes found in phototrophic organisms, and observed their elution order using a Novapak C-18 HPLC column with simple isocratic elution. The elution order of the carotenes was C-30, C-40,C-45 then C-50. The C-40 carotenes with fewer conjugated double bonds (N) had longer retention times. With respect to the end groups, the carotenes eluted in the following order: phi, Psi, epsilon then beta end groups. Furthermore, absorption spectra in the HPLC eluent used were recorded with a photodiode-array detector. A greater N value was associated with a longer absorption maximum wavelength. Since the conjugated end groups (phi and beta) influenced the absorption spectra and the non-conjugated end groups (Psi and epsilon) did not, the number of conjugated end groups (zero, one and two) was clearly distinguishable. Therefore, the chemical structures of carotenes can be easily determined by a combination of the HPLC retention times and the absorption spectra.