M a Dolores Macías-Sµnchez 1 Casimiro Mantell Serrano 1 Miguel Rodríguez Rodríguez 1 Enrique Martínez de la Ossa 1 Luís M. Lubiµn 2 Olimpio Montero 2 1 Department of Chemical Engineering, Food Technology and Environmental Technologies, Science Faculty, University of Cadiz, Cµdiz, Spain 2 Institute for Marine Sciences of Andalucia (CSIC), Cµdiz, Spain Original Paper Extraction of carotenoids and chlorophyll from microalgae with supercritical carbon dioxide and ethanol as cosolvent The extraction of carotenoids and chlorophylls using carbon dioxide modified with ethanol as a cosolvent is an alternative to solvent extraction because it provides a high-speed extraction process. In the study described here, carotenoid and chloro- phyll extraction with supercritical CO 2 + ethanol was explored using freeze-dried powders of three microalgae (Nannochloropsis gaditana, Synechococcus sp. and Dunaliella salina) as the raw materials. The operation conditions were as follows: pressures of 200, 300, 400 and 500 bar, temperatures of 40, 50 and 608C. Analysis of the extracts was performed by measuring the absorbance and by using empirical correlations. The results demonstrate that it is necessary to work at a temperature of 50 – 608C and a pressure range of 300 – 500 bar, depending on the type of microalgae, in order to obtain the highest yield of pigments. The best carotenoid/chlorophyll ratios were obtained by using supercritical fluid extraction + cosolvent instead of using conven- tional extraction. The higher selectivity of the former process should facilitate the separation and purification of the two extracted pigments. Keywords: Carotenoids / Chlorophyll / Cosolvent / Microalgae / Supercritical fluid extraction / Received: October 15, 2007; revised: November 14, 2007; accepted: November 19, 2007 DOI 10.1002/jssc.200700503 1 Introduction Seaweeds have been used as foodstuffs and in agricul- tural applications for a long time. At present, many com- mercial applications of seaweeds are known and these are due to the chemical constitution of these plants [1, 2]. For example, seaweeds are used to increase the nutri- tional content of animal and human foodstuffs, in aqua- culture [3] and in the cosmetics industry [4]. Furthermore, seaweeds have been exploited for about a century as a source of colloids, which find use as thick- eners, gelling agents and stabilisers in the foodstuffs industry. Nevertheless, in the last thirty years seaweeds have been recognised as sources of high added-value compounds. This is the situation with polyunsaturated fatty acids, which are added to children's foodstuffs and nutritional supplements and are also a component of pigments used as natural colourants [5, 6]. Carotenoids or tetraterpenoids are terpenoid pig- ments that contain forty carbon atoms. These materials constitute an important group of natural pigments due to their structural diversity and numerous functions. These compounds play an essential role in photosynthe- sis and for oxygen-consuming life. In microalgae they function as secondary pigments in photosystems, where they work as photo-protectors and as a constituent part of the systems involved in light capture [7 – 9]. Carotenoids are used as colourants in foodstuffs, e.g. they are used as suspensions to give colour to drinks (for instance, b-carotene is used in orange drinks). This col- ourant is unaffected by ascorbic acid, heating or freezing and is active at very low concentrations (1 ppm). Chlorophylls are not used as additives but as protec- tors of natural chlorophylls already present in foodstuffs that are vegetal in origin. Natural chlorophylls are highly unstable and are affected by light, acids or oxygen and their degradation is associated with ageing [10]. These compounds also find use in oils, chewing gums, ice creams, drinks, ready-made soups and in cheeses and yoghurts [11, 12]. Colourant-grade chlorophylls do not have a dose limit for their use, as they are essentially not absorbed by the digestive tract. During technological processes such as scalding or canning of vegetables, heat- ing leads to the disappearance of magnesium from chlor- ophylls. This reaction transforms the chlorophylls into olive green/brown compounds (pheophytins), whose col- Correspondence: Dr. M a Dolores Macías-Sµnchez, Department of Chemical Engineering, Food Technology and Environmental Technologies, Science Faculty, University of Cadiz, Avda. Repfflb- lica Saharaui s/n, 11510 Puerto Real, Cµdiz, Spain E-mail: dolores.macias@uca.es Fax: +34-956016411 i 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com 1352 M. D. Macías-Sµnchez et al. J. Sep. Sci. 2008, 31, 1352 – 1362