times for mixing. Filter and purify to remove contaminating particles and/or air bubbles if necessary.
(b) Mobile phase B—100% EtOAc.
(c) Mobile phase C—100% acetonitrile.
5. HPLC mobile phases for C30 column:
(a) Mobile phase A—100% methanol (MeOH).
(b) Mobile phase B—100% methyl tert-butyl ether (MTBE).
6. An HPLC instrument equipped with a Diode Array Detector
(DAD).
2.3 Fraction
Collection of Individual
Carotenoid Peaks
1. Dark amber-colored HPLC vials, glass inserts, and screw-cap
covers.
2. Sample concentrator with nitrogen gas supply.
3. 100% organic solvent and 100% EtOAc (see Note 4).
4. UV-vis spectrophotometer.
5. Plastic disposable or quartz cuvettes (preferentially) with
10 mm path-length for spectrophotometric measurements.
6. Agilent 1260 Infinity fraction collector or equivalent fraction
collector.
3 Methods
Keeping samples on ice (constant low temperature) and away from
direct high energy light during extraction as well as downstream
analytical steps as a routine practice to avoid carotenoid oxidation
and isomerization. Working with a high number of samples (>50)
is not encouraged for routine carotenoid extraction or HPLC in a
single run due to an increased risk of solvent evaporation and
carotenoid oxidation. When attempting to quantify cis-carotenes
in extracts, 10–20 samples/run are ideal as some carotenes are
highly sensitive to isomerization.
3.1 Plant Tissue
Collection
The collection and handling of plant tissue (sample) is a key step
underpinning the efficiency and accuracy of carotenoid quantification. Carotenoids can undergo isomeric transformation during
tissue maturation [20], and/or in response to changing environmental conditions. Therefore, newly collected plant tissues should
be rapidly snap-frozen after determining the fresh weight of the
sample. Alternatively, the water content can be removed from a
tissue sample using a cold vacuum dryer (4
C or below) and dry
weight measured using a microscale.
For routine analysis of Arabidopsis tissues, we recommend
20–50 mg of green leaves, 75–100 mg of etiolated seedlings, up
148
Yagiz Alagoz et al.
(b) Mobile phase B—100% EtOAc.
(c) Mobile phase C—100% acetonitrile.
5. HPLC mobile phases for C30 column:
(a) Mobile phase A—100% methanol (MeOH).
(b) Mobile phase B—100% methyl tert-butyl ether (MTBE).
6. An HPLC instrument equipped with a Diode Array Detector
(DAD).
2.3 Fraction
Collection of Individual
Carotenoid Peaks
1. Dark amber-colored HPLC vials, glass inserts, and screw-cap
covers.
2. Sample concentrator with nitrogen gas supply.
3. 100% organic solvent and 100% EtOAc (see Note 4).
4. UV-vis spectrophotometer.
5. Plastic disposable or quartz cuvettes (preferentially) with
10 mm path-length for spectrophotometric measurements.
6. Agilent 1260 Infinity fraction collector or equivalent fraction
collector.
3 Methods
Keeping samples on ice (constant low temperature) and away from
direct high energy light during extraction as well as downstream
analytical steps as a routine practice to avoid carotenoid oxidation
and isomerization. Working with a high number of samples (>50)
is not encouraged for routine carotenoid extraction or HPLC in a
single run due to an increased risk of solvent evaporation and
carotenoid oxidation. When attempting to quantify cis-carotenes
in extracts, 10–20 samples/run are ideal as some carotenes are
highly sensitive to isomerization.
3.1 Plant Tissue
Collection
The collection and handling of plant tissue (sample) is a key step
underpinning the efficiency and accuracy of carotenoid quantification. Carotenoids can undergo isomeric transformation during
tissue maturation [20], and/or in response to changing environmental conditions. Therefore, newly collected plant tissues should
be rapidly snap-frozen after determining the fresh weight of the
sample. Alternatively, the water content can be removed from a
tissue sample using a cold vacuum dryer (4
C or below) and dry
weight measured using a microscale.
For routine analysis of Arabidopsis tissues, we recommend
20–50 mg of green leaves, 75–100 mg of etiolated seedlings, up
148
Yagiz Alagoz et al.