8. Collect the upper ethyl acetate phase (~250 μL) enriched in
carotenoid and chlorophyll pigments and transfer to a new
1.5 mL microcentrifuge tube (see Note 7).
9. Centrifuge the upper phase for 5 min, as described in step 7.
10. Transfer approximately 150 μL of the ethyl acetate phase into a
new 1.5 mL microcentrifuge tube. Avoid pipetting near the
bottom of the tube that might have contaminating cellular
debris which can hinder HPLC separation.
11. Transfer 50 μL of the crude extract into an HPLC insert within
a 2 mL amber colored glass vial and secure a sealing cap.
Immediately transfer vials to an HPLC sample collector chamber or temporarily store at À20
C in the dark. Higher temperature and light exposure may cause isomerization. Extracted
samples can be stored at À80
C for several days.
3.3 Carotenoid
Separation:
Comparison
of C18 vs. C30
Columns and Suitable
Solvent Gradients
1. Filter mobile phase solvents to remove oxygen and contaminating particles before filling bottles (1–2 L).
2. Turn on HPLC instrument units and install the appropriate
column. Here, we make reference to the Agilent 1260 Infinity
HPLC system equipped with a Diode Array Detector (DAD)
(see Note 8).
3. Open HPLC instrument software, turn off autosampler compartment illumination and set to 4
C (see Note 9) while
maintaining the column temperature at room temperature
(RT).
4. Load sample vials in the autosampler tray including a blank
(100% EtOAc) in the first position, that help to equilibrate the
column and test for residual pigmentation that could occur
from a previous run if the column was not washed sufficiently.
5. Program the reverse phase gradient method using instrumentation software. We compared the performance of the C18 and
C30 column using reverse-phase solvent gradient as previously
described in [30] and [38], respectively, with only minor
modifications. Separating carotenoid isomers can be difficult
and might require slight modification to gradient programming depending upon the HPLC instrument. We recommend
C18 column chemistry for the analysis of major carotenoids
like Antheraxanthin and Zeaxanthin. However, the C30 column provides a better resolution for the identification of ciscarotene isomers. The selection of the method also depends on
the carotenoid composition of the sample to be analysed.
Figures 1 and 2 compare carotenoid profiles from plants
grown under light and continuous dark, respectively. The composition and reverse-phase solvent gradient used for both columns are as follows:
150
Yagiz Alagoz et al.
carotenoid and chlorophyll pigments and transfer to a new
1.5 mL microcentrifuge tube (see Note 7).
9. Centrifuge the upper phase for 5 min, as described in step 7.
10. Transfer approximately 150 μL of the ethyl acetate phase into a
new 1.5 mL microcentrifuge tube. Avoid pipetting near the
bottom of the tube that might have contaminating cellular
debris which can hinder HPLC separation.
11. Transfer 50 μL of the crude extract into an HPLC insert within
a 2 mL amber colored glass vial and secure a sealing cap.
Immediately transfer vials to an HPLC sample collector chamber or temporarily store at À20
C in the dark. Higher temperature and light exposure may cause isomerization. Extracted
samples can be stored at À80
C for several days.
3.3 Carotenoid
Separation:
Comparison
of C18 vs. C30
Columns and Suitable
Solvent Gradients
1. Filter mobile phase solvents to remove oxygen and contaminating particles before filling bottles (1–2 L).
2. Turn on HPLC instrument units and install the appropriate
column. Here, we make reference to the Agilent 1260 Infinity
HPLC system equipped with a Diode Array Detector (DAD)
(see Note 8).
3. Open HPLC instrument software, turn off autosampler compartment illumination and set to 4
C (see Note 9) while
maintaining the column temperature at room temperature
(RT).
4. Load sample vials in the autosampler tray including a blank
(100% EtOAc) in the first position, that help to equilibrate the
column and test for residual pigmentation that could occur
from a previous run if the column was not washed sufficiently.
5. Program the reverse phase gradient method using instrumentation software. We compared the performance of the C18 and
C30 column using reverse-phase solvent gradient as previously
described in [30] and [38], respectively, with only minor
modifications. Separating carotenoid isomers can be difficult
and might require slight modification to gradient programming depending upon the HPLC instrument. We recommend
C18 column chemistry for the analysis of major carotenoids
like Antheraxanthin and Zeaxanthin. However, the C30 column provides a better resolution for the identification of ciscarotene isomers. The selection of the method also depends on
the carotenoid composition of the sample to be analysed.
Figures 1 and 2 compare carotenoid profiles from plants
grown under light and continuous dark, respectively. The composition and reverse-phase solvent gradient used for both columns are as follows:
150
Yagiz Alagoz et al.