Waters millennium chromatography software

Carotenoids were extracted from 0.5 mg ground tissue samples in a mixture of hexane:acetone:ethanol (2:1:1, v/v/v) as described previously (Tadmor et al., 2005 ), and separated using a Waters 2695 HPLC apparatus equipped with a Waters 996 PDA detector (Milford, MA, USA). Carotenoids were identified by their characteristic absorption spectra, distinctive retention time, and comparison with authentic standards. Quantification was performed by integrating the peak areas with standard curves of authentic standards with the Waters millennium chromatography software. Lutein and β-carotene were relatively quantified at 450 nm and 270 nm, respectively, by integrating their peak areas and calculating their percentage from total integrated peak areas. Tissues for chlorophyll determination were sampled as explained for carotenoid analysis. Chlorophyll extraction was performed in dimmed light to avoid possible photodegradation of chlorophyll. Chlorophyll was extracted by adding 5 ml of DMSO to 0.5 g, vortexing, and incubating in the dark at room temperature for 24 h. The extract was analyzed for absorbance in the wavelengths of 663 nm and 645 nm using a Cary50Bio spectrophotometer (Varian). Chlorophyll concentration was calculated as described by Tadmor et al., (2010) (link).