3.1 Pigment
Extraction
1. Grinding of leaf samples: Precool a mortar and a pestle. Pour a
small amount of liquid N 2 into the mortar and grind
13
Clabeled (or nonlabeled) leaf material (see Note 3) carefully to
fine powder. If you use a ball mill, precool stainless steel balls
and mill adaptors before use. The vibrational frequency and
milling duration should be optimized for different plant leaves.
You can continue with pigment extraction directly, or collect
the frozen leaf powder in a precooled centrifuge tube, freeze it
in liquid N 2 and store at À80
C until extraction.
2. Weigh 15–20 mg of frozen leaf powder quickly; leaf powder
should not thaw while weighing. Write down the exact weight
of each sample (see Note 4).
3. Transfer the weighed frozen leaf powder into a mortar precooled with a small amount of liquid N 2 . Add 1 mL of chilled
acetone and homogenize the sample quickly but thoroughly
until no leaf particles are recognized. Collect the homogenate
into a 2-mL microcentrifuge tube using a pipette (see Note 5)
and put it on ice.
4. Rinse the mortar and pestle with 0.5 mL of chilled acetone. Do
not discard this acetone; it should be collected and added to the
sample. Adjust the total volume of the sample to 2 mL. The
optimal volume for extraction is different for different plant
leaves.
5. Repeat steps 2–4 for all samples.
6. Centrifuge the samples at 15,700 Â g, 4
C for 5 min.
7. Collect the green supernatant into a fresh microcentrifuge tube
and put it on ice. Check the color of the pellets remaining at the
bottom of the first microcentrifuge tubes. If the pellets are still
greenish or yellowish, extraction is not complete. Repeat the
steps 3, 4, and 6 until the pellets become white (see Note 6).
Table 1
Chemical formulae and monoisotopic mass of photosynthetic carotenoids in plants
Carotenoid
Abbreviation
Chemical formula
Monoisotopic mass
a [u]
Violaxanthin
Vio
C 40 H 56 O 4
600.417869
Neoxanthin
Neo
C 40 H 56 O 4
600.417869
Antheraxanthin
Anth
C 40 H 56 O 3
584.42295
Lutein
Lut
C 40 H 56 O 2
568.42803
Zeaxanthin
Zea
C 40 H 56 O 2
568.42803
α-Carotene
α-Car
C 40 H 56
536.43820
β-Carotene
β-Car
C 40 H 56
536.43820
a
Values are for the lightest (most abundant) isotopologs
266
Bjo ¨ rn Thiele and Shizue Matsubara
Extraction
1. Grinding of leaf samples: Precool a mortar and a pestle. Pour a
small amount of liquid N 2 into the mortar and grind
13
Clabeled (or nonlabeled) leaf material (see Note 3) carefully to
fine powder. If you use a ball mill, precool stainless steel balls
and mill adaptors before use. The vibrational frequency and
milling duration should be optimized for different plant leaves.
You can continue with pigment extraction directly, or collect
the frozen leaf powder in a precooled centrifuge tube, freeze it
in liquid N 2 and store at À80
C until extraction.
2. Weigh 15–20 mg of frozen leaf powder quickly; leaf powder
should not thaw while weighing. Write down the exact weight
of each sample (see Note 4).
3. Transfer the weighed frozen leaf powder into a mortar precooled with a small amount of liquid N 2 . Add 1 mL of chilled
acetone and homogenize the sample quickly but thoroughly
until no leaf particles are recognized. Collect the homogenate
into a 2-mL microcentrifuge tube using a pipette (see Note 5)
and put it on ice.
4. Rinse the mortar and pestle with 0.5 mL of chilled acetone. Do
not discard this acetone; it should be collected and added to the
sample. Adjust the total volume of the sample to 2 mL. The
optimal volume for extraction is different for different plant
leaves.
5. Repeat steps 2–4 for all samples.
6. Centrifuge the samples at 15,700 Â g, 4
C for 5 min.
7. Collect the green supernatant into a fresh microcentrifuge tube
and put it on ice. Check the color of the pellets remaining at the
bottom of the first microcentrifuge tubes. If the pellets are still
greenish or yellowish, extraction is not complete. Repeat the
steps 3, 4, and 6 until the pellets become white (see Note 6).
Table 1
Chemical formulae and monoisotopic mass of photosynthetic carotenoids in plants
Carotenoid
Abbreviation
Chemical formula
Monoisotopic mass
a [u]
Violaxanthin
Vio
C 40 H 56 O 4
600.417869
Neoxanthin
Neo
C 40 H 56 O 4
600.417869
Antheraxanthin
Anth
C 40 H 56 O 3
584.42295
Lutein
Lut
C 40 H 56 O 2
568.42803
Zeaxanthin
Zea
C 40 H 56 O 2
568.42803
α-Carotene
α-Car
C 40 H 56
536.43820
β-Carotene
β-Car
C 40 H 56
536.43820
a
Values are for the lightest (most abundant) isotopologs
266
Bjo ¨ rn Thiele and Shizue Matsubara