individual carotenoids based upon their individual light absorption
properties and unique spectral conformations, which in some cases
isoforms can be difficult to distinguish within a crude carotenoid
extract mixture without clear separation by retention time.
Here we describe a rapid and simple carotenoid extraction
protocol that is suitable for both photosynthetic and nonphotosynthetic plant tissues, and compare two solvent gradient-based HPLC
methods using C18 and C30 reverse-phase column chemistry to
separate cis/trans carotenoids. We also describe how to identify
carotenoids and their isoforms as well as develop standards for
absolute quantification.
2 Materials
Use of HPLC-grade (or analytical) reagents and ultrapure deionized water is recommended. For health and safety concerns, it is
advised to comply with associated risk assessments, standard
operating procedures, and waste disposal protocols.
2.1 Crude Extraction
of Carotenoids from
Plants
1. Extraction buffer: Acetone–ethyl acetate (EtOAc) (6:4, v/v).
For 1 L extraction buffer, mix 600 mL of 100% acetone with
400 mL of 100% EtOAc then add and dissolve 1 g butylated
hydroxytoluene (BHT) in the mixture (see Note 1).
2. 100% ultrapure H 2 O (to be used as a phase separation buffer).
3. 1.5 and 2 mL microcentrifuge tubes and micropipettes with
suitable tips.
4. Liquid nitrogen and ice to be used while/after grinding and to
store ground plant tissue samples.
5. Stainless steel beads or pestle and mortar for mechanical grinding of plant tissues (see Note 2).
6. A tissue grinder or cell lysis system with appropriate adapters
(see Note 3).
7. Refrigerated centrifuge, vortexer, and/or mixers.
2.2 Reverse-Phase
HPLC-Based
Quantification
of Carotenoids
1. Dark amber colored HPLC vials (2 mL), glass inserts (0.2 mL)
and screw cap 8 mm open with silicone/polytetrafluoroethylene (PTFE) septa.
2. C18 HPLC column: C18 (250 Â 4.6, 4-μm).
3. C30 HPLC column: C30 (250 Â 4.6 mm, S-5 μm).
4. HPLC mobile phases for C18 column:
(a) Mobile phase A—acetonitrile–H 2 O–triethylamine (TEA)
(9:1:0.01, v/v/v). For 1 L mobile phase A, mix 900 mL
of 100% acetonitrile, 100 mL of ultrapure H 2 O and 1 mL
TEA in a 1 L glass bottle. Gently invert the bottle 2–3
Profiling Carotenoids in Plants Using HPLC
147
properties and unique spectral conformations, which in some cases
isoforms can be difficult to distinguish within a crude carotenoid
extract mixture without clear separation by retention time.
Here we describe a rapid and simple carotenoid extraction
protocol that is suitable for both photosynthetic and nonphotosynthetic plant tissues, and compare two solvent gradient-based HPLC
methods using C18 and C30 reverse-phase column chemistry to
separate cis/trans carotenoids. We also describe how to identify
carotenoids and their isoforms as well as develop standards for
absolute quantification.
2 Materials
Use of HPLC-grade (or analytical) reagents and ultrapure deionized water is recommended. For health and safety concerns, it is
advised to comply with associated risk assessments, standard
operating procedures, and waste disposal protocols.
2.1 Crude Extraction
of Carotenoids from
Plants
1. Extraction buffer: Acetone–ethyl acetate (EtOAc) (6:4, v/v).
For 1 L extraction buffer, mix 600 mL of 100% acetone with
400 mL of 100% EtOAc then add and dissolve 1 g butylated
hydroxytoluene (BHT) in the mixture (see Note 1).
2. 100% ultrapure H 2 O (to be used as a phase separation buffer).
3. 1.5 and 2 mL microcentrifuge tubes and micropipettes with
suitable tips.
4. Liquid nitrogen and ice to be used while/after grinding and to
store ground plant tissue samples.
5. Stainless steel beads or pestle and mortar for mechanical grinding of plant tissues (see Note 2).
6. A tissue grinder or cell lysis system with appropriate adapters
(see Note 3).
7. Refrigerated centrifuge, vortexer, and/or mixers.
2.2 Reverse-Phase
HPLC-Based
Quantification
of Carotenoids
1. Dark amber colored HPLC vials (2 mL), glass inserts (0.2 mL)
and screw cap 8 mm open with silicone/polytetrafluoroethylene (PTFE) septa.
2. C18 HPLC column: C18 (250 Â 4.6, 4-μm).
3. C30 HPLC column: C30 (250 Â 4.6 mm, S-5 μm).
4. HPLC mobile phases for C18 column:
(a) Mobile phase A—acetonitrile–H 2 O–triethylamine (TEA)
(9:1:0.01, v/v/v). For 1 L mobile phase A, mix 900 mL
of 100% acetonitrile, 100 mL of ultrapure H 2 O and 1 mL
TEA in a 1 L glass bottle. Gently invert the bottle 2–3
Profiling Carotenoids in Plants Using HPLC
147