![]() ![]() However, HPLC is also expensive both in terms of time and instrumentation. HPLC can resolve most chlorophylls and carotenoids, including their degradation products such as pheophytins and pheophorbides, as long as relevant pigment standards are available. High performance liquid chromatography (HPLC) is considered the “gold standard” for measuring pigment concentrations in plant and algal samples. Pigment composition is also an important physiological response parameter, because the relative pigment concentrations are influenced by environmental factors such as light and nutrient availability. Because pigment composition can be a reflection of taxonomic composition, presence or absence of certain marker pigments can be used to identify phytoplankton community composition. The relative concentrations of other photosynthetic and photo-protective pigments can provide valuable taxonomical and physiological information. Chlorophyll a (chl a) concentrations, for example, are widely used by plankton ecologists as a proxy for phytoplankton biomass and for estimating primary productivity. ![]() Quantification of phytoplankton pigments is an integral part of inland water monitoring and general experimental research involving phytoplankton. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedĭata Availability: All relevent data and scripts are contained within the Supporting Information of this paper.įunding: This project was funded in part by internal grants from Norwegian Institute for Water Research for SH, MG, MK, and TR, and by internal grants from University of Oslo and Norwegian University of Life Sciences for JET, MK, and TR. Received: FebruAccepted: AugPublished: September 11, 2015Ĭopyright: © 2015 Thrane et al. (2015) Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution. 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.Ĭitation: Thrane J-E, Kyle M, Striebel M, Haande S, Grung M, Rohrlack T, et al. 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. 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. 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). 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. The fitted background is shown to be higher than a solvent blank, with features reflecting contributions from both scatter and non-pigment absorption. 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. We here present several improvements for this type of methodology, including adaptation to plate reader technology and efficient model fitting by open source software. 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. ![]()
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