Fast, Sensitive, and Inexpensive Alternative to Analytical Pigment HPLC:  Quantification of Chlorophylls and Carotenoids in Crude Extracts by Fitting with Gauss Peak Spectra

Quantification of pigments in complex mixtures is an important task in the physiology of photosynthetic organisms, because pigment composition differs depending on the species, tissue, and physiological state. Currently available methods, however, are either limited to very few pigments (classical UV/vis spectroscopic methods), or they are time-consuming, labor intensive, or costly (e.g., HPLC). Here we describe a UV/vis spectrophotometric method that is capable of a rapid (∼1 min/sample) and inexpensive (<1 euro/sample) quantification of more than a dozen pigments in a crude extract, which means it is suitable for high-throughput screening applications. A detection limit of <1 pmol for each pigment allows for determining the pigment composition in only 0.5 μg of lyophilized leaves or algae. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these “Gauss peak spectra” including an automatic correction of wavelength inaccuracy, baseline instability, sample turbidity, and effects of temperature/water content. Here we present the Gauss peak spectra from 350 to 750 nm for acetone solutions of all chlorophyll and carotenoid derivatives that are abundant (including conditions of Cd, Cu, or Zn stress) in leaves of higher plants, Euglena, brown algae, and various cyanobacteria like Anabaena and Trichodesmium:  [Mg]-Chl a, b, c1, c2; pheophytin a, b; [Cd]-Chl a, b; [Cu]-Chl a, b; [Zn]-Chl a, b; antheraxanthin, aurochrome, β-carotene, β-cryptoxanthin, cis- and trans-canthaxanthin, diadinochrome (=diadinoxanthin 5,6-epoxide), cis- and trans-diadinoxanthin, diatoxanthin, cis- and trans-echinenone, fucoxanthin, lutein, myxoxanthophyll, neoxanthin, violaxanthin, and all three stereoisomers of zeaxanthin in acetone. We present extensive tests of our new quantification method for determining optimal and limiting conditions of its performance and for comparison with previous methods. Finally, we show application examples for Thlaspi fendleri (Chlorophyta), Euglena gracilisc (Euglenophyta), Ectocarpus siliculosus (Phaeophyta), and Trichodesmium erythraeum IMS101 (cyanobacteria).