7 Analytical Protocols in Carotenoid Analysis
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processes (Halim et al. 2013). Microwave is another technology applied for disruption of cells that reduces the amount of solvent required for extraction, but caution
must be considered because of the heat that may cause degradation (Pasquetet al.
2011). Finally, the reader should notice that though modern processing technologies
considerably reduce the amount of solvent required for efficient extraction, they still
need revision of several key operative conditions (Kim et al. 2016), and that their
use is not intended for the lab scale.
Once the pre-treatment process has been performed, extraction with organic
solvent(s) is the next step where other critical factors should be considered. Selection of solvent or solvents mixture is made according to the frequently wide polarity
range of the carotenoid profile. Hence, the occurrence of polar xanthophylls, and
apolar carotenes and xanthophyll esters in the cellular contents demands the use of
solvents combinations made with hexane, diethyl ether, ethanol, acetone, and others
(Alfonsi et al. 2008; Soares et al. 2016). For industrial processing of biomass, other
factors are considered such as sustainability and eco-friendly production, as well as
health and safety issues. At the lab, ethyl acetate either alone or in combination with
apolar solvents (hexane) fulfills the wide polarity range requirements for successful
extraction of carotenoids (Strati and Oreopoulou 2011). Solvent mixtures from the
protocols designed by Folch et al. (1957), Bligh and Dyer (1959), and Christie (1993)
are equally recommended for lipid extraction from tissues including microalgae
(Axelsson and Gentili 2014). These protocols are based in solvent mixtures of chloroform, methanol, and water, while the solvent to sample ratio could be adjusted
considering the effectivity of the pre-treatment process applied for cell disruption.
Supercritical fluid extraction is a technology designed for extraction of valuable
compounds from different raw materials including microalgae (Liau et al. 2010; Patil
et al. 2013). Although its application is aimed at the semi-pilot and industrial scales,
it is interesting for the reader to have a brief summary of this eco-friendly technique
for extraction. The driving force of the supercritical fluid extraction is the application
of high pressure to convert a gas (CO 2 ) into a fluid that still shows a viscosity as
gas. In these conditions, the fluid penetrates the raw material achieving a high efficiency of mass transfer in a short time. Although supercritical CO 2 is an apolar fluid,
this feature can be tailored by adding co-solvents (ethanol) that widen the polarity
range of the extraction. Other conditions that are extremely favorable for carotenoid
extraction are the low temperature conditions and the short time of the operation,
while the final extract is completely free of extractant fluid as CO 2 backs to its gas
state at normal pressure conditions (Zaghdoudi et al. 2016). Indeed, optimization of
these conditions may produce a selectivity of the extracted compounds from the raw
material and research activities have been focused in this aim (Macías-Sánchez et al.
2007; Kitada et al. 2009; Guedes et al. 2013; Goto et al. 2015; Reyes et al. 2016). This
possibility points to the supercritical fluid extraction as a technology that potentially
yields extracts with a high purity of the selected target compounds from microalgae.
Another advantage is that the extraction procedure could be coupled online with the
analytical technique, liquid or gas chromatography and even supercritical fluid chromatography, so that the extract is immediately analyzed and after data interpretation,
the information could be feedbacked to optimize extraction conditions, allowing a
155
processes (Halim et al. 2013). Microwave is another technology applied for disruption of cells that reduces the amount of solvent required for extraction, but caution
must be considered because of the heat that may cause degradation (Pasquetet al.
2011). Finally, the reader should notice that though modern processing technologies
considerably reduce the amount of solvent required for efficient extraction, they still
need revision of several key operative conditions (Kim et al. 2016), and that their
use is not intended for the lab scale.
Once the pre-treatment process has been performed, extraction with organic
solvent(s) is the next step where other critical factors should be considered. Selection of solvent or solvents mixture is made according to the frequently wide polarity
range of the carotenoid profile. Hence, the occurrence of polar xanthophylls, and
apolar carotenes and xanthophyll esters in the cellular contents demands the use of
solvents combinations made with hexane, diethyl ether, ethanol, acetone, and others
(Alfonsi et al. 2008; Soares et al. 2016). For industrial processing of biomass, other
factors are considered such as sustainability and eco-friendly production, as well as
health and safety issues. At the lab, ethyl acetate either alone or in combination with
apolar solvents (hexane) fulfills the wide polarity range requirements for successful
extraction of carotenoids (Strati and Oreopoulou 2011). Solvent mixtures from the
protocols designed by Folch et al. (1957), Bligh and Dyer (1959), and Christie (1993)
are equally recommended for lipid extraction from tissues including microalgae
(Axelsson and Gentili 2014). These protocols are based in solvent mixtures of chloroform, methanol, and water, while the solvent to sample ratio could be adjusted
considering the effectivity of the pre-treatment process applied for cell disruption.
Supercritical fluid extraction is a technology designed for extraction of valuable
compounds from different raw materials including microalgae (Liau et al. 2010; Patil
et al. 2013). Although its application is aimed at the semi-pilot and industrial scales,
it is interesting for the reader to have a brief summary of this eco-friendly technique
for extraction. The driving force of the supercritical fluid extraction is the application
of high pressure to convert a gas (CO 2 ) into a fluid that still shows a viscosity as
gas. In these conditions, the fluid penetrates the raw material achieving a high efficiency of mass transfer in a short time. Although supercritical CO 2 is an apolar fluid,
this feature can be tailored by adding co-solvents (ethanol) that widen the polarity
range of the extraction. Other conditions that are extremely favorable for carotenoid
extraction are the low temperature conditions and the short time of the operation,
while the final extract is completely free of extractant fluid as CO 2 backs to its gas
state at normal pressure conditions (Zaghdoudi et al. 2016). Indeed, optimization of
these conditions may produce a selectivity of the extracted compounds from the raw
material and research activities have been focused in this aim (Macías-Sánchez et al.
2007; Kitada et al. 2009; Guedes et al. 2013; Goto et al. 2015; Reyes et al. 2016). This
possibility points to the supercritical fluid extraction as a technology that potentially
yields extracts with a high purity of the selected target compounds from microalgae.
Another advantage is that the extraction procedure could be coupled online with the
analytical technique, liquid or gas chromatography and even supercritical fluid chromatography, so that the extract is immediately analyzed and after data interpretation,
the information could be feedbacked to optimize extraction conditions, allowing a