13. The organic phase frequently reduces its volume due to the
high volatility of diethyl ether. If this happens the original
diethyl ether volume should be approximately restored with
more solvent before proceeding to next step.
14. If some water residues are still present after solvent evaporation, add a few milliliters (3–5 mL) of absolute ethanol and
evaporate again in the rotary evaporator. The addition of ethanol will facilitate the evaporation of water by forming an
azeotrope.
15. Introduce the sample into a sonication bath during 10 s for
ensuring solubilization of carotenoids.
16. If carotenoids are not analyzed immediately after the extraction, samples should be stored at À30
C (or lower) until
chromatographic analysis (HPLC) in order to prevent degradation of carotenoids. If possible, the air from the sample vial
should be displaced with a stream of N 2 .
17. A preliminary test sample should be analyzed for an adequate
selection of sample size.
18. A heating thermo-block for tubes at temperature <30
C can
be used to speed up the process.
19. In the case of the extract prepared by the “Microscale procedure” (Subheading 3.4), the entire extract can be submitted to
saponification.
20. A single phase should be obtained. In the event that two phases
are separated, add a few drops of methanol until a single phase
is formed, and mix thoroughly.
21. Alternatively, samples can be filtered through 0.45 μm nylon
filters. However, this practice is not recommended when using
volatile solvents as evaporation during extract manipulation
will cause overestimation of carotenoid content due to sample
concentration.
22. Samples which have been kept at low temperature in the freezer
should be left at room temperature for 5–10 min to ensure
solubilization of pigments. For the same reason these samples
cannot be centrifuged right out of the freezer.
23. If available, the injection should be achieved with an automatic
injector for better reproducibility and automatization. In addition the sample compartment of the automatic injector should
be fitted with a cooling system for sample vials in order to
prevent thermo-degradation as well as sample concentration
due to solvent evaporation. The recommended temperature
range is 10–15
C. Lower temperature should not be used as
carotenoid can precipitate in the vial thus leading to errors in
the quantification.
HPLC Analysis of Carotenoids
131
high volatility of diethyl ether. If this happens the original
diethyl ether volume should be approximately restored with
more solvent before proceeding to next step.
14. If some water residues are still present after solvent evaporation, add a few milliliters (3–5 mL) of absolute ethanol and
evaporate again in the rotary evaporator. The addition of ethanol will facilitate the evaporation of water by forming an
azeotrope.
15. Introduce the sample into a sonication bath during 10 s for
ensuring solubilization of carotenoids.
16. If carotenoids are not analyzed immediately after the extraction, samples should be stored at À30
C (or lower) until
chromatographic analysis (HPLC) in order to prevent degradation of carotenoids. If possible, the air from the sample vial
should be displaced with a stream of N 2 .
17. A preliminary test sample should be analyzed for an adequate
selection of sample size.
18. A heating thermo-block for tubes at temperature <30
C can
be used to speed up the process.
19. In the case of the extract prepared by the “Microscale procedure” (Subheading 3.4), the entire extract can be submitted to
saponification.
20. A single phase should be obtained. In the event that two phases
are separated, add a few drops of methanol until a single phase
is formed, and mix thoroughly.
21. Alternatively, samples can be filtered through 0.45 μm nylon
filters. However, this practice is not recommended when using
volatile solvents as evaporation during extract manipulation
will cause overestimation of carotenoid content due to sample
concentration.
22. Samples which have been kept at low temperature in the freezer
should be left at room temperature for 5–10 min to ensure
solubilization of pigments. For the same reason these samples
cannot be centrifuged right out of the freezer.
23. If available, the injection should be achieved with an automatic
injector for better reproducibility and automatization. In addition the sample compartment of the automatic injector should
be fitted with a cooling system for sample vials in order to
prevent thermo-degradation as well as sample concentration
due to solvent evaporation. The recommended temperature
range is 10–15
C. Lower temperature should not be used as
carotenoid can precipitate in the vial thus leading to errors in
the quantification.
HPLC Analysis of Carotenoids
131