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A. Pérez-Gálvez and J. Fontecha
high-throughput screening. The features of this online coupling are detailed in the
chromatographic analysis section.
Finally, a common derivatization process applied to the extract is chemical hydrolysis with ethanolic or methanolic KOH, in order to hydrolyze the xanthophyll esters
to the corresponding free forms, reduce the content in triacylglycerides that interfere
in the subsequent chromatographic analysis as well as the chlorophyll pigments,
which are transformed to acyclic degradation products. Chemical hydrolysis is not
suitable for screening the presence of xanthophyll esters and the determination of
chlorophyll and carotenoid pigments in the same measurement.
To summarize this topic, extraction with solvent is the choice for lab processing
of microalgae samples while the application of cold pre-treatment for disruption
of cells is always recommended. In our laboratory the routinely applied protocol
consists of filtering an aliquot of the biomass using a 47 mm glass fiber filter (Zhang
et al. 2016; Maroneze et al. 2019). The filter containing the biomass is weighed and
immediately froze at −80 °C. When it is grinded in a mortar with liquid nitrogen, the
crushed filter material significantly increases the solid shear forces, while the liquid
nitrogen releases any heat produced by the mechanical process and contributes to
the disruption process. Subsequently, the powder is mixed with 10 mL of extraction
solvent (9:1, N,N-dimethylformamide:water) under continuous stirring for 15 min
at 4 °C, and then centrifuged (10,000 rpm, 4 min). The organic layer is accumulated
in a decanting funnel and the solid residue is re-extracted with 10 mL hexane mixed
in an ultrasonic bath (5 min, 720 W), vortexed (5 min), and after the addition of
10 mL NaCl solution (10% w/v) the mixture is centrifuged (10,000 rpm, 5 min).
The supernatant is added to the funnel and the pellet is re-suspended with 10 mL
diethyl ether, mixed in an ultrasonic bath (5 min, 720 W), vortexed (5 min), and
finally centrifuged (10,000 rpm, 5 min) after the addition of 10 mL NaCl solution
(10% w/v). The combined solvent fractions in the funnel are extracted with diethyl
ether and NaCl solution (10% w/v). The upper phase is isolated and concentrated to
dryness in a rotary evaporator. The residue is dissolved in acetone and stored at −
20 °C.
7.2.2 Spectroscopic Measurement
The use of spectrophotometry as a simple, fast, and low-cost technique for quantification of the pigment content in extracts is still a suitable analytical procedure
recommended for some experiments. Hence, phytoplankton pigments in samples
from sediments, natural lake aliquots, pure, or single species cultures can be spectrophotometrically monitored to estimate productivity and physiological and toxicological studies, as these measurements do not usually require an exact resolution
of the pigment content (Kobayashi et al. 1993; Sairam et al. 2002; Marschall and
Proctor 2004). Spectrophotometric methods are available in several fashions considering alternatives of solvents for the measurement and equations for determination
A. Pérez-Gálvez and J. Fontecha
high-throughput screening. The features of this online coupling are detailed in the
chromatographic analysis section.
Finally, a common derivatization process applied to the extract is chemical hydrolysis with ethanolic or methanolic KOH, in order to hydrolyze the xanthophyll esters
to the corresponding free forms, reduce the content in triacylglycerides that interfere
in the subsequent chromatographic analysis as well as the chlorophyll pigments,
which are transformed to acyclic degradation products. Chemical hydrolysis is not
suitable for screening the presence of xanthophyll esters and the determination of
chlorophyll and carotenoid pigments in the same measurement.
To summarize this topic, extraction with solvent is the choice for lab processing
of microalgae samples while the application of cold pre-treatment for disruption
of cells is always recommended. In our laboratory the routinely applied protocol
consists of filtering an aliquot of the biomass using a 47 mm glass fiber filter (Zhang
et al. 2016; Maroneze et al. 2019). The filter containing the biomass is weighed and
immediately froze at −80 °C. When it is grinded in a mortar with liquid nitrogen, the
crushed filter material significantly increases the solid shear forces, while the liquid
nitrogen releases any heat produced by the mechanical process and contributes to
the disruption process. Subsequently, the powder is mixed with 10 mL of extraction
solvent (9:1, N,N-dimethylformamide:water) under continuous stirring for 15 min
at 4 °C, and then centrifuged (10,000 rpm, 4 min). The organic layer is accumulated
in a decanting funnel and the solid residue is re-extracted with 10 mL hexane mixed
in an ultrasonic bath (5 min, 720 W), vortexed (5 min), and after the addition of
10 mL NaCl solution (10% w/v) the mixture is centrifuged (10,000 rpm, 5 min).
The supernatant is added to the funnel and the pellet is re-suspended with 10 mL
diethyl ether, mixed in an ultrasonic bath (5 min, 720 W), vortexed (5 min), and
finally centrifuged (10,000 rpm, 5 min) after the addition of 10 mL NaCl solution
(10% w/v). The combined solvent fractions in the funnel are extracted with diethyl
ether and NaCl solution (10% w/v). The upper phase is isolated and concentrated to
dryness in a rotary evaporator. The residue is dissolved in acetone and stored at −
20 °C.
7.2.2 Spectroscopic Measurement
The use of spectrophotometry as a simple, fast, and low-cost technique for quantification of the pigment content in extracts is still a suitable analytical procedure
recommended for some experiments. Hence, phytoplankton pigments in samples
from sediments, natural lake aliquots, pure, or single species cultures can be spectrophotometrically monitored to estimate productivity and physiological and toxicological studies, as these measurements do not usually require an exact resolution
of the pigment content (Kobayashi et al. 1993; Sairam et al. 2002; Marschall and
Proctor 2004). Spectrophotometric methods are available in several fashions considering alternatives of solvents for the measurement and equations for determination