Development of a standardized methodology for quantifying total chlorophyll and carotenoids from foliage of hardwood and conifer tree species

Despite the availability of several protocols for the extraction of chlorophylls and carotenoids from foliage of forest trees, information regarding their respective extraction efficiencies is scarce. We compared the efficiencies of acetone, ethanol, dimethyl sulfoxide (DMSO), and N, N-dimethylformamide (DMF) over a range of incubation times for the extraction of chlorophylls and carotenoids using small amounts of unmacerated tissue. Of the 11 species studied, comparable amounts of chlorophyll were extracted by all four solvents from three species and by ethanol and DMF from nine species. In four species, acetone, ethanol, and DMF extracted comparable chlorophyll amounts, while in another two species comparable amounts were extracted by ethanol, DMSO, and DMF. In one species, ethanol extracted significantly greater amounts of chlorophyll compared with all other solvents. The brown coloration of DMSO extracts for some species compromised the calculations of chlorophylls and carotenoids, making DMSO a poor choice. Overall, extraction efficiencies of ethanol and DMF were comparable for analyzing chlorophyll concentrations. However, because DMF is more toxic than ethanol, we recommend ethanol as the better option of these two for chlorophyll extractions. On the other hand, DMF is the most efficient solvent among the four tested for the extraction of carotenoids from these species. The results presented will facilitate the design of multispecies local- and regional-scale ecological studies to evaluate forest health. Additionally, they will enable reliable comparisons of results from multiple laboratories and (or) studies that used different solvents and help validate chlorophyll estimates obtained by remote sensing.