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Application of compressed fluid–based extraction and purification procedures to obtain astaxanthin-enriched extracts from Haematococcus pluvialis and characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry


The green microalga Haematococcus pluvialis has been widely studied due to its capacity to accumulate great amounts of astaxanthin, a high-value carotenoid with biological activities. In the present work, two green compressed fluid–based processes, pressurized liquid extraction (PLE) and supercritical antisolvent fractionation (SAF), are integrated to obtain an astaxanthin-enriched extract from this microalga. PLE was carried out using pressurized ethanol as solvent, for 20 min, at 10 MPa, and 50 °C as extraction temperature. Subsequently, the obtained extract was processed by SAF to further purify the carotenoid fraction. The SAF process was optimized using a 3-level factorial experimental design and considering three experimental variables: (i) CO2 pressure (10–30 MPa), (ii) percentage of water in the PLE extract (20–50%), and (iii) PLE extract/supercritical-CO2 flow rate ratio (0.0125–0.05). Total carotenoid content was evaluated in both extracts and raffinates. Best results were obtained at 30 MPa, 0.05 feed/SC-CO2 mass flow rate, and 20% (v/v) of water in the feed solution, achieving values of 120.3 mg g−1 carotenoids in extract (in the SAF extract fraction), which were significantly higher than those obtained in the original PLE extract. In parallel, a new fast two-dimensional comprehensive liquid chromatography (LC×LC) method was optimized to get the full carotenoid profile of these extracts in less than 25 min. This is the first time that the use of a C30 column is reported in an on-line LC×LC system.

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The authors thank Microphyt (Baillargues, France) for kindly providing H. pluvialis biomass.


The authors would like to thank projects ABACUS (Algae for a Biomass Applied to the production of added value compounds—funded by the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 745668) and AGL2017-89417-R (MINECO, Spain) for funding.

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Correspondence to Miguel Herrero.

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Gallego, R., Arena, K., Dugo, P. et al. Application of compressed fluid–based extraction and purification procedures to obtain astaxanthin-enriched extracts from Haematococcus pluvialis and characterization by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry. Anal Bioanal Chem 412, 589–599 (2020).

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  • Supercritical antisolvent fractionation
  • SAF
  • Haematococcus pluvialis
  • PLE
  • Astaxanthin
  • LC×LC