(a) C18 column: Separate carotenoids using C18-Mobile
phase A and C18-Mobile phase B with the following
gradient: 0 min 100% A (1 mL/min); 3 min 100% A
(1 mL/min); 31 min 33.3% A and 66.7% B (1 mL/
min); 31.2 min 100% B (1.5 mL/min); 32 min 100% B
(1.5 mL/min), 35 min 100% A (1 mL/min).
(b) C30 column: Separate carotenoids using C30-Mobile
phase A and C30-Mobile phase B with the following
gradient: 0 min 100% A; 5 min 100% A; 25 min 100% B;
30 min 100% A, 35 min (or until column equilibration is
achieved depending upon isomers detected) 100% A with
the flow rate of 1 mL/min.
6. Adjust the DAD detection range to record signals at 286 nm,
348 nm, 400 nm, 440 nm, 454 nm, and 470 nm (see Note 13).
7. Start HPLC instrument software to inject 20–50 μL of pigment extract through the HPLC column and facilitate carotenoid separation as per programmed gradient (see Note 10).
Xanthophylls
C30
mAU (440nm)
WT-3W-Y.leaf
䣯䣫䣰
8
10
12
14
16
18
20
0
100
200
300
400
500
600
Vio
9c-Vio
Neo
Chl-b
Lut
Chl-a
βc
βc-2
βc-2
βc-c1
Ant
cis-carotenes
䣯䣫䣰
28
crtiso-7DAS-etio_cot.
ziso-7DAS-etio_cot.
18
20
22
24
26
0
20
40
60
80
100
120
140
160
Neu
isomers
Lyc
isomers
C30
mAU (440nm)
tc-ζcar
ζcar1;2
dc-ζcar
P-Neu
P-Lyc
䣯䣫䣰
24
25
26
27
28
29
30
31
0
10
20
30
40
50
60
70
80
crtiso-7DAS-etio_cot.
ziso-7DAS-etio_cot.
C18
mAU (440nm)
tc-ζcar
dc-ζcar
P-Neu
P-Lyc
7,9dc-Lyc
32
βc
Vio
Neo
Ant
Lut
Chl-b
Chl-a
䣯䣫䣰
5
10
15
20
25
30
0
20
40
60
80
100
120
mAU (440nm)
C18
WT-3W-Y.leaf
0
Overlapping
peaks
Overlapping
peaks
Zea
Zea
(crtiso only)
(crtiso only)
(crtiso only)
(crtiso only)
crtiso-3W-Y.leaf
crtiso-3W-Y.leaf
Fig. 1 Distribution of xanthophylls and carotenes on C18 vs. C30 columns. Tissue samples for HPLC were
collected from 3-week-old young Arabidopsis leaves and 7-day-old dark-grown cotyledons of Arabidopsis ziso
(zic1-3) and crtiso (ccr2.1) mutants. Abbreviations: 3W, 3-week-old; cot, cotyledon; etio, etiolated; mAU, milliAbsorbance Units; Y.leaf, young leaf. For the full nomenclature of carotenoid abbreviations; see Table 1
Profiling Carotenoids in Plants Using HPLC
151
phase A and C18-Mobile phase B with the following
gradient: 0 min 100% A (1 mL/min); 3 min 100% A
(1 mL/min); 31 min 33.3% A and 66.7% B (1 mL/
min); 31.2 min 100% B (1.5 mL/min); 32 min 100% B
(1.5 mL/min), 35 min 100% A (1 mL/min).
(b) C30 column: Separate carotenoids using C30-Mobile
phase A and C30-Mobile phase B with the following
gradient: 0 min 100% A; 5 min 100% A; 25 min 100% B;
30 min 100% A, 35 min (or until column equilibration is
achieved depending upon isomers detected) 100% A with
the flow rate of 1 mL/min.
6. Adjust the DAD detection range to record signals at 286 nm,
348 nm, 400 nm, 440 nm, 454 nm, and 470 nm (see Note 13).
7. Start HPLC instrument software to inject 20–50 μL of pigment extract through the HPLC column and facilitate carotenoid separation as per programmed gradient (see Note 10).
Xanthophylls
C30
mAU (440nm)
WT-3W-Y.leaf
䣯䣫䣰
8
10
12
14
16
18
20
0
100
200
300
400
500
600
Vio
9c-Vio
Neo
Chl-b
Lut
Chl-a
βc
βc-2
βc-2
βc-c1
Ant
cis-carotenes
䣯䣫䣰
28
crtiso-7DAS-etio_cot.
ziso-7DAS-etio_cot.
18
20
22
24
26
0
20
40
60
80
100
120
140
160
Neu
isomers
Lyc
isomers
C30
mAU (440nm)
tc-ζcar
ζcar1;2
dc-ζcar
P-Neu
P-Lyc
䣯䣫䣰
24
25
26
27
28
29
30
31
0
10
20
30
40
50
60
70
80
crtiso-7DAS-etio_cot.
ziso-7DAS-etio_cot.
C18
mAU (440nm)
tc-ζcar
dc-ζcar
P-Neu
P-Lyc
7,9dc-Lyc
32
βc
Vio
Neo
Ant
Lut
Chl-b
Chl-a
䣯䣫䣰
5
10
15
20
25
30
0
20
40
60
80
100
120
mAU (440nm)
C18
WT-3W-Y.leaf
0
Overlapping
peaks
Overlapping
peaks
Zea
Zea
(crtiso only)
(crtiso only)
(crtiso only)
(crtiso only)
crtiso-3W-Y.leaf
crtiso-3W-Y.leaf
Fig. 1 Distribution of xanthophylls and carotenes on C18 vs. C30 columns. Tissue samples for HPLC were
collected from 3-week-old young Arabidopsis leaves and 7-day-old dark-grown cotyledons of Arabidopsis ziso
(zic1-3) and crtiso (ccr2.1) mutants. Abbreviations: 3W, 3-week-old; cot, cotyledon; etio, etiolated; mAU, milliAbsorbance Units; Y.leaf, young leaf. For the full nomenclature of carotenoid abbreviations; see Table 1
Profiling Carotenoids in Plants Using HPLC
151