material without getting stuck at the bottom of the tube. For
samples like roots, fruits, and large seeds, we recommend using
a pestle and mortar. The 2 mL tubes are ideal in case 1 mL
extraction buffer is particularly needed where the required
tissue weight is more than 50 mg for extraction.
3. The tissue grinder adapters should be prechilled in a À80
C
freezer for at least 1 h prior to tissue grinding. Do not snapfreeze in liquid nitrogen as it causes damage.
4. There are multiple options available to be used as organic
solvents like ethanol, benzene, acetone, petroleum, and chloroform. Keep in mind that a specific absorption coefficient
(A
1%
1cm ) is unique to that particular organic solvent used to
calculate the absolute quantities after spectrophotometric measurements. An expanded list of carotenoid absorption coefficients for various solvents can be found elsewhere [39–41].
5. The tissue samples from mutant lines that hyper-accumulate
cis-carotenes (like dark-grown etiolated cotyledon tissues)
Table 2
Identification of carotenoid isomers with C18 column in light-grown Arabidopsis leaves and darkgrown cotyledons of Arabidopsis ziso and crtiso mutants
Carotenoid (C18)
RT (min)
λ (nm)
Chemical formula
Neoxanthin (Neo)
9.84
414, 438, 467
C 40 H 56 O 4
Violaxanthin (Vio)
12.22
418, 442, 471
C 40 H 56 O 4
Antheraxanthin (Ant)
15.48
422, 447, 477
C 40 H 56 O 3
Lutein (Lut)
17.92
422, 448, 476
C 40 H 56 O 2
All-trans-zeaxanthin (Zea)
18.6
430, 450, 480
C 40 H 56 O 2
Chlorophyll-B (major peak) (Chl-b)
22.04
_, 458, _
C 55 H 70 MgN 4 O 6
Chlorophyll-A (major peak) (Chl-a)
24.53
_, 430, _
C 55 H 72 MgN 4 O 5
β-carotene (βc)
30.82
428, 456, 482
C 40 H 56
7,9-di-cis-lycopene (dc-Lyc)
29.53
442, 468
C 40 H 56
Tetra-cis-lycopene (P-Lyc)
30.03
420, 442, 468
C 40 H 56
Tri-cis-neurosporene (P-Neu)
30.95
388, 410, 435, 464
C 40 H 58
Tri-cis-ζ-carotene (tc-ζc)
31.37
380, 400, 425
C 40 H 60
di-cis-ζ-carotene (dc-ζc)
31.63
382, 402, 428
C 40 H 60
Phytofluene isomer-1 (Phf-1)
31.98
332, 349, 368
C 40 H 62
Phytofluene isomer-2 (Phf-2)
32.2
332, 350, 368
C 40 H 62
Phytoene (Phy)
32.64
_, 286, _
C 40 H 64
Profiling Carotenoids in Plants Using HPLC
157
samples like roots, fruits, and large seeds, we recommend using
a pestle and mortar. The 2 mL tubes are ideal in case 1 mL
extraction buffer is particularly needed where the required
tissue weight is more than 50 mg for extraction.
3. The tissue grinder adapters should be prechilled in a À80
C
freezer for at least 1 h prior to tissue grinding. Do not snapfreeze in liquid nitrogen as it causes damage.
4. There are multiple options available to be used as organic
solvents like ethanol, benzene, acetone, petroleum, and chloroform. Keep in mind that a specific absorption coefficient
(A
1%
1cm ) is unique to that particular organic solvent used to
calculate the absolute quantities after spectrophotometric measurements. An expanded list of carotenoid absorption coefficients for various solvents can be found elsewhere [39–41].
5. The tissue samples from mutant lines that hyper-accumulate
cis-carotenes (like dark-grown etiolated cotyledon tissues)
Table 2
Identification of carotenoid isomers with C18 column in light-grown Arabidopsis leaves and darkgrown cotyledons of Arabidopsis ziso and crtiso mutants
Carotenoid (C18)
RT (min)
λ (nm)
Chemical formula
Neoxanthin (Neo)
9.84
414, 438, 467
C 40 H 56 O 4
Violaxanthin (Vio)
12.22
418, 442, 471
C 40 H 56 O 4
Antheraxanthin (Ant)
15.48
422, 447, 477
C 40 H 56 O 3
Lutein (Lut)
17.92
422, 448, 476
C 40 H 56 O 2
All-trans-zeaxanthin (Zea)
18.6
430, 450, 480
C 40 H 56 O 2
Chlorophyll-B (major peak) (Chl-b)
22.04
_, 458, _
C 55 H 70 MgN 4 O 6
Chlorophyll-A (major peak) (Chl-a)
24.53
_, 430, _
C 55 H 72 MgN 4 O 5
β-carotene (βc)
30.82
428, 456, 482
C 40 H 56
7,9-di-cis-lycopene (dc-Lyc)
29.53
442, 468
C 40 H 56
Tetra-cis-lycopene (P-Lyc)
30.03
420, 442, 468
C 40 H 56
Tri-cis-neurosporene (P-Neu)
30.95
388, 410, 435, 464
C 40 H 58
Tri-cis-ζ-carotene (tc-ζc)
31.37
380, 400, 425
C 40 H 60
di-cis-ζ-carotene (dc-ζc)
31.63
382, 402, 428
C 40 H 60
Phytofluene isomer-1 (Phf-1)
31.98
332, 349, 368
C 40 H 62
Phytofluene isomer-2 (Phf-2)
32.2
332, 350, 368
C 40 H 62
Phytoene (Phy)
32.64
_, 286, _
C 40 H 64
Profiling Carotenoids in Plants Using HPLC
157