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

Abstract

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 (approximately 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 microg 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, beta-carotene, beta-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).

Complexation and Toxicity of Copper in Higher Plants. II. Different Mechanisms for Copper versus Cadmium Detoxification in the Copper-Sensitive Cadmium/Zinc Hyperaccumulator Thlaspi caerulescens (Ganges Ecotype)

“…For pigment quantification, leaves were frozen in liquid nitrogen, then lyophilized, and finally extracted in 100% acetone. Chls and carotenoids were quantified according to Kü pper et al (2007c) as described in detail in our companion article (Kü pper et al, 2009) on C. helmsii.…”

Complexation and Toxicity of Copper in Higher Plants. I. Characterization of Copper Accumulation, Speciation, and Toxicity inCrassula helmsiias a New Copper Accumulator

“…Spectra of pigment extracts were measured with the UV/visible spectrophotometer Lambda 16 (Perkin-Elmer) at a spectral bandwidth of 1 nm with 0.2-nm sampling interval from 350 to 750 nm. Chls were quantified according to the "Gauss-peak-spectra" (GPS) method developed by Kü pper et al (2000bKü pper et al ( , 2007b. Normalized spectra (350-750 nm) of pigment standards were mathematically described by a series of Gaussian peaks.…”

“…The combinations of GPS were then fitted to the sample spectrum to obtain the parameters representing the concentrations of the individual components. Additionally, baseline drift and wavelength inaccuracies of the spectrophotometer as well as temperature differences, residual water content, and turbidity of the samples were automatically corrected (Kü pper et al, 2007b).…”

Abiotic modulation of Spartina maritima photobiology in different latitudinal populations

“…Extraction occurred at À20 C during 24 h in the dark to prevent pigment degradation. After extraction, samples were centrifuged at 4000 rpm for 15 min at 4 C. For pigment analysis the GPS method was used (Kupper et al, 2007). Samples were scanned in a dual beam spectrophotometer from 350 nm to 750 nm at 0.5 nm steps.…”