should be collected under nonphotosynthetic dim green LED
light to prevent carotenoid photo-oxidation and
photoisomerisation.
6. Ground tissue powder can stick to the edges of the microcentrifuge lid following grinding. Gently tap the tubes several
times before opening to transfer tissue to the bottom of the
vial. Ensure no liquid N 2 enters the microcentrifuge tube as this
can result in an explosion after sealing the tube.
7. Carotenoids are positioned within the upper ethyl acetate
phase, so the acetone–EtOAc ratio can be changed (e.g., 4:6,
v/v) if a higher volume of extract is needed. Consider any
changes to this dilution when calculating the carotenoid abundances in samples.
8. Before performing HPLC analysis, consider using a guard
column integrated between the injector and the analytical column (C18 & C30). Guard columns protect the analytical
column against blockage that can arise due to the presence of
impurities inside the crude extract and or solvents if not filtered
in advance.
9. A longer HPLC run (~10 h or longer) when the auto-sampler
compartment is set to 4
C can cause condensation of water in
the compartment. It is preferable to maintain the auto-sampler
compartment at 6–7
C when analysing large numbers of
samples in a single run.
10. We recommend running HPLC with 100% mobile phase B
prior to sample injection until the back pressure stabilizes.
11. Pure carotenoid standards can be readily purchased and used to
validate retention times and carotenoid spectra.
12. Calculation of % III/II ratio can facilitate the characterization
of spectral fine structures in identified peaks. The %III/II value
is dependent to the organic solvents being used and can be
calculated by dividing the maximum absorption unit (λ max ) of
peak III by λ max of peak II by taking the minimum absorption
value in-between as a baseline (λ baseline ), multiplied by
100 ([λ maxIII Àλ baseline ]/[λ maxII Àλ baseline ] Â 100). The λ maxIII,
λ maxII and λ baseline can be calculated as shown in Fig. 5.
13. Due to their different spectral characteristics, carotenoid abundance should be quantified by taking specific measurements at
the maximum appropriate wavelength. It is recommended to
use absorbance values taken at 440 nm for the majority of
carotenoids except for phytoene (at 286 nm), phytofluene
(at 348 nm), ζ-carotene (at 400 nm), β-carotene
(at 454 nm), and lycopene isomers (at 470 nm) excluding
7,9,9
0 ,7
0 -tetra-cis-lycopene (prolycopene) (at 440 nm).
Profiling Carotenoids in Plants Using HPLC
159
light to prevent carotenoid photo-oxidation and
photoisomerisation.
6. Ground tissue powder can stick to the edges of the microcentrifuge lid following grinding. Gently tap the tubes several
times before opening to transfer tissue to the bottom of the
vial. Ensure no liquid N 2 enters the microcentrifuge tube as this
can result in an explosion after sealing the tube.
7. Carotenoids are positioned within the upper ethyl acetate
phase, so the acetone–EtOAc ratio can be changed (e.g., 4:6,
v/v) if a higher volume of extract is needed. Consider any
changes to this dilution when calculating the carotenoid abundances in samples.
8. Before performing HPLC analysis, consider using a guard
column integrated between the injector and the analytical column (C18 & C30). Guard columns protect the analytical
column against blockage that can arise due to the presence of
impurities inside the crude extract and or solvents if not filtered
in advance.
9. A longer HPLC run (~10 h or longer) when the auto-sampler
compartment is set to 4
C can cause condensation of water in
the compartment. It is preferable to maintain the auto-sampler
compartment at 6–7
C when analysing large numbers of
samples in a single run.
10. We recommend running HPLC with 100% mobile phase B
prior to sample injection until the back pressure stabilizes.
11. Pure carotenoid standards can be readily purchased and used to
validate retention times and carotenoid spectra.
12. Calculation of % III/II ratio can facilitate the characterization
of spectral fine structures in identified peaks. The %III/II value
is dependent to the organic solvents being used and can be
calculated by dividing the maximum absorption unit (λ max ) of
peak III by λ max of peak II by taking the minimum absorption
value in-between as a baseline (λ baseline ), multiplied by
100 ([λ maxIII Àλ baseline ]/[λ maxII Àλ baseline ] Â 100). The λ maxIII,
λ maxII and λ baseline can be calculated as shown in Fig. 5.
13. Due to their different spectral characteristics, carotenoid abundance should be quantified by taking specific measurements at
the maximum appropriate wavelength. It is recommended to
use absorbance values taken at 440 nm for the majority of
carotenoids except for phytoene (at 286 nm), phytofluene
(at 348 nm), ζ-carotene (at 400 nm), β-carotene
(at 454 nm), and lycopene isomers (at 470 nm) excluding
7,9,9
0 ,7
0 -tetra-cis-lycopene (prolycopene) (at 440 nm).
Profiling Carotenoids in Plants Using HPLC
159