Carotenoids are typically measured in tissues with the high performance liquid chromatography (HPLC) and quantitation is usually done by calibrating with stock solutions in solvents. Four carotenoids including lutein, zeaxanthin, lycopene and β-carotene were dissolved in hexane and methanol respectively, and their absorbance characteristics were compared. Lutein shows absorbance spectra that are almost independent of solvents at various concentrations. Spectra of zeaxanthin, lycopene and β-carotene were found to be more solvent-dependent. The absorbance of zeaxanthin at λ(max) is about ~2 times larger in methanol than in hexane at the higher concentrations, and increased non-linearly with increasing concentration in hexane. The absorbance of lycopene at λ(max) in hexane is ~4 fold larger than in methanol, but the absorbance of the methanol sample can be recovered by re- extracting this sample in hexane. The absorbance of β-carotene in hexane is larger than in methanol, and increased linearly with increasing concentration. But β-carotene showed a non-linear concentration effect in methanol. There are very small variations in λ(max) for all four carotenoids between hexane and methanol, due to differences in molar extinction coefficients. The non-linear concentration effects for these carotenoids are probably due to differences in solubility leading to the formation of microcrystals. Thus, care should be taken with quantitation of tissue carotenoid values, when they depend on measurement of concentrations in stock solutions.
- UV-Visible spectroscopy
- β- Carotene