7. Analyze 2 μl of each external standard sample by HPLC and
determine the α-tocopheryl acetate peak area A EST .
8. In order to calculate phytoene amounts in the samples, use the
following conversions:
(a) First, convert peak area of phytoene A Phyt virtually into
β-carotene, which the HPLC was calibrated for, by multiplication with factor 1.97. This factor originates from
molar extinction coefficients ε for β-carotene
(134,000 L Â mol
À1
 cm
À1 ) and phytoene
(68,125 L Â mol
À1
 cm
À1
) [23]. Second, compensate
for sample losses during extraction as well as fluctuations
in HPLC injection volumes as follows. Calculate the mean
of all three A EST (A EST_MEAN ) and divide A EST_MEAN by
A IST for every individual sample to determine a samplespecific tocopheryl acetate (TocAc) correction factor.
Third, divide the converted phytoene peak area A Phyt by
the TocAc correction factor. In summary, this provides the
corrected phytoene peak area A PhytCORR (¼ A Phyt  1.97/
TocAc correction factor) (see Note 14).
(b) Convert corrected phytoene peak area A PhytCORR into
pmol of phytoene (amount Phyt ) by division by the
β-carotene response factor RF car . This yields the amountPhyt [pmol] in the 2 μl injected from your sample. Multiply
by factor 50 (¼ 100 μl extract sample/2 μl injection
volume) to determine the total phytoene amount in your
100 μl extract sample [pmol], that is, amount Phyt_tot .
(c) Convert amount Phyt_tot into total mass of phytoene
[ng] using the molecular weight of phytoene of
545 g mol
À1 . Further divide this by the sample’s dry
weight to determine total mass of phytoene per dry
weight [ng phytoene mg
À1 DW) (see Note 15).
4 Notes
1. HPLC system with photodiode array detector (e.g., our Shimadzu HPLC system consisting of degasser DGU-20A3, two
pumps LC-20AD XR, autosampler SIL-20 AC XR, thermostatted column compartment CTO-20AC, photodiode array
detector SPD-M20A, and system controller unit CBM-20A
(Shimadzu, Duisburg, Germany)).
2. Plant health has great impact on carotenoid flux. Use nonsenescent, healthy leaves for experiments. If several different lines
are compared, grow them simultaneously under same conditions and make sure plant health and leaf quality is comparable.
We usually use leaves from Arabidopsis grown at long day
conditions.
288
Julian Koschmieder and Ralf Welsch
determine the α-tocopheryl acetate peak area A EST .
8. In order to calculate phytoene amounts in the samples, use the
following conversions:
(a) First, convert peak area of phytoene A Phyt virtually into
β-carotene, which the HPLC was calibrated for, by multiplication with factor 1.97. This factor originates from
molar extinction coefficients ε for β-carotene
(134,000 L Â mol
À1
 cm
À1 ) and phytoene
(68,125 L Â mol
À1
 cm
À1
) [23]. Second, compensate
for sample losses during extraction as well as fluctuations
in HPLC injection volumes as follows. Calculate the mean
of all three A EST (A EST_MEAN ) and divide A EST_MEAN by
A IST for every individual sample to determine a samplespecific tocopheryl acetate (TocAc) correction factor.
Third, divide the converted phytoene peak area A Phyt by
the TocAc correction factor. In summary, this provides the
corrected phytoene peak area A PhytCORR (¼ A Phyt  1.97/
TocAc correction factor) (see Note 14).
(b) Convert corrected phytoene peak area A PhytCORR into
pmol of phytoene (amount Phyt ) by division by the
β-carotene response factor RF car . This yields the amountPhyt [pmol] in the 2 μl injected from your sample. Multiply
by factor 50 (¼ 100 μl extract sample/2 μl injection
volume) to determine the total phytoene amount in your
100 μl extract sample [pmol], that is, amount Phyt_tot .
(c) Convert amount Phyt_tot into total mass of phytoene
[ng] using the molecular weight of phytoene of
545 g mol
À1 . Further divide this by the sample’s dry
weight to determine total mass of phytoene per dry
weight [ng phytoene mg
À1 DW) (see Note 15).
4 Notes
1. HPLC system with photodiode array detector (e.g., our Shimadzu HPLC system consisting of degasser DGU-20A3, two
pumps LC-20AD XR, autosampler SIL-20 AC XR, thermostatted column compartment CTO-20AC, photodiode array
detector SPD-M20A, and system controller unit CBM-20A
(Shimadzu, Duisburg, Germany)).
2. Plant health has great impact on carotenoid flux. Use nonsenescent, healthy leaves for experiments. If several different lines
are compared, grow them simultaneously under same conditions and make sure plant health and leaf quality is comparable.
We usually use leaves from Arabidopsis grown at long day
conditions.
288
Julian Koschmieder and Ralf Welsch