Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution
The Gauss-peak spectra (GPS) method represents individual pigment spectra as weighted sums of Gaussian functions, and uses these to model absorbance spectra of phytoplankton pigment mixtures. We here present several improvements for this type of methodology, including adaptation to plate reader technology and efficient model fitting by open source software. We use a one-step modeling of both pigment absorption and background attenuation with non-negative least squares, following a one-time instrument-specific calibration. The fitted background is shown to be higher than a solvent blank, with features reflecting contributions from both scatter and non-pigment absorption. We assessed pigment aliasing due to absorption spectra similarity by Monte Carlo simulation, and used this information to select a robust set of identifiable pigments that are also expected to be common in natural samples. To test the method’s performance, we analyzed absorbance spectra of pigment extracts from sediment cores, 75 natural lake samples, and four phytoplankton cultures, and compared the estimated pigment concentrations with concentrations obtained using high performance liquid chromatography (HPLC). The deviance between observed and fitted spectra was generally very low, indicating that measured spectra could successfully be reconstructed as weighted sums of pigment and background components. Concentrations of total chlorophylls and total carotenoids could accurately be estimated for both sediment and lake samples, but individual pigment concentrations (especially carotenoids) proved difficult to resolve due to similarity between their absorbance spectra. In general, our modified-GPS method provides an improvement of the GPS method that is a fast, inexpensive, and high-throughput alternative for screening of pigment composition in samples of phytoplankton material.
Department of Biosciences, Centre for Ecological and Evolutionary Synthesis (CEES), University of Oslo, Oslo, Norway;Department of Biosciences, Section for Aquatic Biology and Toxicology (AQUA), University of Oslo, Oslo, Norway;Department of Environmental Sciences, Norwegian University of Life Sciences (NMBU), Ås, Norway;Institute for Chemistry and Biology of the Marine Environment (ICBM), Carl-von-Ossietzky University Oldenburg, Oldenburg, Germany;Norwegian Institute for Water Research (NIVA), Oslo, Norway;Norwegian Institute for Water Research (NIVA), Oslo, Norway;Department of Biosciences, Section for Aquatic Biology and Toxicology (AQUA), University of Oslo, Oslo, Norway;Department of Environmental Sciences, Norwegian University of Life Sciences (NMBU), Ås, Norway;Department of Biosciences, Section for Aquatic Biology and Toxicology (AQUA), University of Oslo, Oslo, Norway
Recommended Citation:
Jan-Erik Thrane,Marcia Kyle,Maren Striebel,et al. Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution[J]. PLOS ONE,2015-01-01,10(9)