Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution

PLoS One. 2015 Sep 11;10(9):e0137645. doi: 10.1371/journal.pone.0137645. eCollection 2015.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • High-Throughput Screening Assays / methods*
  • Microalgae / chemistry
  • Pigments, Biological / chemistry*
  • Plant Extracts / chemistry*
  • Spectrophotometry / methods

Substances

  • Pigments, Biological
  • Plant Extracts

Grants and funding

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. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.