High-throughput methods for the analysis of pigments in aquatic sediments

High-throughput methods for the analysis of complex mixtures of sedimentary pigments have been developed by improving the efficiency and speed of pigment extraction and chromatographic analysis. A method for the routine and automated extraction of highly sensitive chlorin pigments from sediments has been developed and compared with the widely used method of sonic extraction. Pigments were identified by high performance liquid chromatography with on-line photodiode array detection and multistage tandem mass spectrometry. The efficacy and integrity of accelerated solvent extraction (ASE) varies for temperatures within the range 20oC to 175oC. The pigment yields and absence of alteration products indicate 70oC to be the optimal temperature for ASE in pigment analysis, yields and distributions of pigments being very similar to those obtained using sonication. Extraction time and solvent consumption are considerably lower than those for sonication, leading to increased efficiency and lower environmental impact and cost. Significant improvements in precision for quantification were achieved by freeze drying and sieving sediments prior to extraction. In addition, an ultra-high performance liquid chromatography (UHPLC) method has been developed for the analysis of complex pigment distributions in natural samples, examining the effects of, and optimising, flow rate, injection volume, column length, mobile phase composition and column temperature. The new chromatographic methods have been demonstrated for the analysis of pigment compositions in an extract from sediment (Priest Pot, Cumbria UK) and a natural water-column filtrand (Little Long Lake). Detection and identification of components was achieved by online UV/Vis detector and multistage tandem mass spectrometry (up to MS3). The separation, identification and confirmation by MS of photosynthetic pigments from Priest Pot and bacteriophaeophytin c and d homologues from Little Long Lake is achieved in under 18 min, five times faster than the established HPLC method. The new ASE and UHPLC methods were applied to the study of three sediment cores from Lake Heidi, Lake Nella and Lake 14, Larsemann Hills, Antarctica at high sampling resolution. The methods offer significant savings in time, solvent and sample consumption, have improved limits of detection and generate more reliable data. Hence, the use of the methods will enable the rapid screening of environmental samples and the profiling of sediment cores at high sampling resolutions within reasonable timescales.