Keywords

Carotenoids are widely distributed in all living organisms including higher plants, algae, bacteria, and yeast. They are insoluble in water, but are fat-soluble and contain conjugated double bond systems. Carotenoids are classified into two major groups, the carotenes and the xanthophylls. Carotenes are hydrocarbons without oxygen and are few in number, such as α-, β-, γ-, δ-, ε-, ζ-carotene, and lycopene. Xanthophylls contain at least one atom of oxygen in the molecule and make up the vast majority of the carotenoids. Presently, over 700 carotenoids have been identified. Carotenoids act as accessory light-harvesting pigments, effectively extending the range of light absorbed by the photosynthetic apparatus, and perform an essential photoprotective role by quenching triplet state chlorophyll molecules and scavenging singlet oxygen and other toxic oxygen species formed within the chloroplast. Fucoxanthin, peridinin, and siphonaxanthin are class-specific carotenoids in light-harvesting complexes and work as efficient antenna pigments harvesting blue-to-green light (Rowan 1989).

1 Distribution of Carotenoids in the Algal Class

Over 40 kinds of xanthophylls and five carotenes have been found in the various algal species, as summarized in Table 13.1. Different algal phyla have distinct carotenoid profiles. Some carotenoids are found only in some algal divisions or classes; therefore, these carotenoids can be used as chemotaxonomic markers (Goodwin 1974; Rowan 1989; van den Hoek et al. 1995; Takaichi 2011; Huang et al. 2017).

Table 13.1 The distribution of carotenoids in the algal classes and absorption maxima of these carotenoids
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2 Carotenoid Analysis by HPLC

There are many methods for the analysis of photosynthetic pigments, including classical paper chromatography and thin layer chromatography (TLC). The simple TLC method has been used for the rapid separation of pigments of algae. The separation is carried out on plates coated with adsorbents, such as silica gel, cellulose, Al2O3, or polyamide. Colored spots separated by TLC are scraped off the plates and extracted in a small funnel fitted with a sintered glass filter. The light absorption spectrum of the compound may thus be determined. As for more quantitative and accurate methods, high performance liquid chromatography (HPLC) has been applied to the analysis of algal pigments in recent decades (Wright et al. 1991; Wang et al. 1999; Hu 2003). HPLC has made it possible to simultaneously determine the concentration of a wide range of carotenoids and chlorophylls and their degradation products. Although numerous HPLC techniques have been published to date, pigment separations are typically conducted with a reverse-phase C18 column and a mobile phase of methanol, acetone, ammonium acetate, and ethyl acetate (Schmid and Stich 1995). HPLC-separated pigment peaks are routinely identified by comparison of retention time values with those of standards and on-line diode array UV/Vis spectroscopy at 440 nm.

In reversed-phase chromatography, substances eluting at the solvent front are considered to be polar, such as chlorophyllide b, chlorophyllide a, and chlorophyll c (peak 1 in Fig. 13.1). Peridinin is the most polar carotenoid (peak 2 of Peridinium pusillum in Fig. 13.1), then siphonaxanthin, fucoxanthin, violaxanthin (peak 3), diadinoxanthin (peak 4 of P. pusillum), diatoxanthin (peak 8) and lutein are eluted in order of decreasing polarity. At the non-polar end of the chromatogram, there are chlorophyll b, chlorophyll a (peak 9), pheophytin b, pheophytin a, and carotenes (Wright et al. 1991). The retention time values of peaks 4 in P. pusillum and Nannochloropsis sp. are the same (Fig. 13.1), but the latter carotenoids is violaxanthin-like based on its absorption spectrum. Therefore, to identify a pigment, both retention time and absorption spectrum should be considered. Pigment quantification is based on the linear relationship between the weight of standard injected and the resulting peak area.

Fig. 13.1
figure 1

The HPLC chromatogram (440 nm) of pigments in Peridinium pusillum and Nannochloropsis sp.

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3 Quantification of Total Carotenoids

Since carotenoids are sensitive to oxygen, heat, light, and acids, all operations should be performed in dim light or in total darkness and at low temperature.

  1. 1.

    Extraction: Centrifuge the fresh culture, add acetone or methanol, ultrasonicate (for macroalgae, grind samples gently) and transfer the extract to a new tube, repeat extraction until no more colored material can be extracted.

  2. 2.

    Purification: Extracts might contain chlorophylls and can be removed by saponification. Saponification is performed in a small volume of ether and an equal volume of 10% methanolic KOH solution is added. The saponification takes place at room temperature for 1–2 h in the dark, then water is added (containing 5% NaCl) to a separatory funnel, and the ether layer is separated from the aqueous phase. The latter is extracted several times with more ether and the combined extracts washed three times with water to remove alkali and methanol. The content of carotenoids is estimated from the maximum absorbancy measured in the 450 nm region as follows (Jensen 1978):

$$ C=\left(D\times V\times f\times 10\right)/2500 $$

The total amount of carotenoids is then C (in milligrams), the absorbancy D in a 1.0 cm cell, the volume (milliliters) of the original extract is V, and the dilution factor is f. It is assumed that the pigments have an average extinction coefficient of 2500.

4 Note

HPLC can be used as a qualitative and quantitative analysis, while spectrophotometry can only be used for measuring the total carotenoid content. Relatively speaking, the former method is more accurate.