Absorption spectra and extinction coefficients of the crystalline astaxanthin (Fig. 1) and astacene (Fig. 2), were determined in acetone, benzene, pyridine, carbon disulfide, petroleum ether, n-hexane and cyclo-hexane (Figs. 3, 4 and Table 1). Astaxanthin in petroleum ether was chromatographically almost pure, but in seldom cases separated into two fractions (Ax. -Fr. A and B), whereas astacene usually separated into two fractions (Ac. -Fr. A and B). On the observations of absorption spectra of these fractions, we assumed that Ax.- and Ac. -Fr. B correspond to mono-cis isomer of Ax.- and Ac.-Fr. A respectively, which are all-trans form and commonly called “astaxanthin” and “astacene”.
Using the crystalline pigment mentioned above as authentic samples, identification of the muscle carotenoid of the North Pacific salmon (Oncorhynchus nerka, O. kisutch, O. gorbuscha, O. tschawytscha, and O. keta) was performed. The muscle was extracted with acetone and the pigment was transfered to petroleum ether, and chromatographed on cellulose powder to remove non-pigment (Fr. -1) lipids which were eluted without being absorbed. The pigement was easily removed from the column with acetone (Fr. -2). Fr. -1 contained on carotenoids. Fr. -2 was partitioned between petroleum ether and 90% aqueous methanol. It was all hypophasic. And then Fr. -2 was chromatographed on CaCO₃. The results obtained show that the muscle of these salmon contained astaxanthin as its main fraction (Fr. -2-1) and small amounts of the fraction (Fr. -2-2) which was considered as mono-cis isomer of astaxanthin. The amount of the latter was about one-tenth of the former. The latter pigment is perhaps the artifacts produced during the working up process such as chromatography, and etc.. Astacene and β-carotene were not identified. The same results were obtained in chromatography using alumina as absorbent.