17. Add another 2 ml PE:DE (2:1; v:v) to sample and repeat steps
13–15 to repartition.
18. Transfer upper organic phase to 15 ml tube and dry combined
organic phases in a vacuum evaporator or under a nitrogen
stream.
19. Transfer extract into 2 ml tube using 2 Â 100 μl chloroform/
methanol (2:1, v/v).
20. Dry organic phase using a SpeedVac.
21. Dissolve dried extract in exactly 100 μl of chloroform/methanol (2:1, v/v), vortex.
22. Transfer complete extract into a HPLC vial and inject 2 μl for
HPLC analysis (see Subheading 3.5).
3.5 Phytoene
Quantification by HPLC
Analysis
The quantification procedure described here for phytoene can in
principle be applied for the quantification of all carotenoids if
chromatographic conditions allow for sufficient resolution of analytes and if molar extinction coefficients are known.
1. Separation of samples is performed on a C18 column (e.g.,
Hypersil Gold C18 UPLC-column; 150 Â 2.1 mm i.d.,
1.9 μm, Thermo Scientific), using the solvent system A, 0.1%
formic acid in water and B, 0.1% formic acid in acetonitrile. The
mobile gradient at a constant flow rate of 0.5 mL min
À1 and at
20
C is as follows: 70% B for 1 min, linear increase to 100% B
within 4 min and separation at 100% B for 20 min. The column
is then reequilibrated to 70% B (see Note 12).
2. Prepare β-carotene solution of about 5 pmol/μl in petroleum
ether (see Note 13).
3. Analyze 1, 2, 5, 10, 25, and 50 pmol β-carotene on column by
HPLC. Use the same injection volume in each run (e.g., 2 μl
from 50 μl β-carotene solutions of appropriate concentration).
Perform all concentrations in independent triplicates.
4. Integrate the β-carotene peak area in a chromatogram at
450 nm and plot the peak areas for all measurements against
the amount of β-carotene on column. Generate a calibration
curve to infer the ratio β-carotene area per pmol β-carotene,
that is, the response factor for β-carotene (RF car ).
5. Analyze 2 μl of plant extracts (total volume 100 μl, see Subheading 3.4) by HPLC.
6. Identify phytoene and the internal standard α-tocopheryl acetate using a chromatogram at 285 nm which is λ max for both
analytes (see Fig. 1 for norflurazon-treated leaves and Fig. 2 for
norflurazon-treated calli). Determine the phytoene peak area
A Phyt and the α-tocopheryl acetate peak area A IST.
286
Julian Koschmieder and Ralf Welsch
13–15 to repartition.
18. Transfer upper organic phase to 15 ml tube and dry combined
organic phases in a vacuum evaporator or under a nitrogen
stream.
19. Transfer extract into 2 ml tube using 2 Â 100 μl chloroform/
methanol (2:1, v/v).
20. Dry organic phase using a SpeedVac.
21. Dissolve dried extract in exactly 100 μl of chloroform/methanol (2:1, v/v), vortex.
22. Transfer complete extract into a HPLC vial and inject 2 μl for
HPLC analysis (see Subheading 3.5).
3.5 Phytoene
Quantification by HPLC
Analysis
The quantification procedure described here for phytoene can in
principle be applied for the quantification of all carotenoids if
chromatographic conditions allow for sufficient resolution of analytes and if molar extinction coefficients are known.
1. Separation of samples is performed on a C18 column (e.g.,
Hypersil Gold C18 UPLC-column; 150 Â 2.1 mm i.d.,
1.9 μm, Thermo Scientific), using the solvent system A, 0.1%
formic acid in water and B, 0.1% formic acid in acetonitrile. The
mobile gradient at a constant flow rate of 0.5 mL min
À1 and at
20
C is as follows: 70% B for 1 min, linear increase to 100% B
within 4 min and separation at 100% B for 20 min. The column
is then reequilibrated to 70% B (see Note 12).
2. Prepare β-carotene solution of about 5 pmol/μl in petroleum
ether (see Note 13).
3. Analyze 1, 2, 5, 10, 25, and 50 pmol β-carotene on column by
HPLC. Use the same injection volume in each run (e.g., 2 μl
from 50 μl β-carotene solutions of appropriate concentration).
Perform all concentrations in independent triplicates.
4. Integrate the β-carotene peak area in a chromatogram at
450 nm and plot the peak areas for all measurements against
the amount of β-carotene on column. Generate a calibration
curve to infer the ratio β-carotene area per pmol β-carotene,
that is, the response factor for β-carotene (RF car ).
5. Analyze 2 μl of plant extracts (total volume 100 μl, see Subheading 3.4) by HPLC.
6. Identify phytoene and the internal standard α-tocopheryl acetate using a chromatogram at 285 nm which is λ max for both
analytes (see Fig. 1 for norflurazon-treated leaves and Fig. 2 for
norflurazon-treated calli). Determine the phytoene peak area
A Phyt and the α-tocopheryl acetate peak area A IST.
286
Julian Koschmieder and Ralf Welsch