10. Collect the ether phases and evaporate to dryness under vacuum in a rotary evaporator at temperature not higher than
30
C. Alternatively, solvent can be evaporated under N 2
stream.
11. Dissolve the dry extract in 0.5–1.0 mL of acetone (see
Note 15) and stored at À30
C until HPLC analysis.
12. Extraction should be performed in at least triplicate.
3.4 Saponification
If saponification is required for the hydrolysis of lipids and carotenoid esters, the following micro-scale procedure can be used.
1. 1 mL of the pigment extract obtained in Subheading 3.2
(step 10) is placed in a 15 mL centrifuge tube (Falcon type
or similar) and dried under a nitrogen stream (see Note 19).
2. Add 2 mL of diethyl ether and 1 mL of 10% (w/v) KOH–
methanol.
3. Shake the mixture in a vortex for 30 s and left to react for
20 min at room temperature under nitrogen atmosphere and
with periodic shaking (see Note 20).
4. Add 8 mL of distilled water for washing, and separate phases by
centrifugation (at 4500 Â g at 4
C during 5 min).
5. Discard the lower aqueous phase and repeat the washing operation (see Note 13) until the pH of the aqueous phase is neutral
(use pH test strips).
6. Discard the lower phase (last washing).
7. Filter the ether phase through an anhydrous Na 2 SO 4 , and rinse
the filtering material with more diethyl ether. Collect the filtrate in a clean test tube.
8. Evaporate the ether to dryness under a nitrogen stream.
9. Dissolve the extract in 1 mL of acetone and store at À30
C
until subsequent chromatographic analysis.
3.5 High
Performance Liquid
Chromatography
(HPLC)
1. Equilibrate the HPLC equipment to the initial solvent conditions (75% solvent A and 25% solvent B) and 25
C for column
temperature.
2. Set flow rate at 1 mL/min.
3. Prior to injection, samples are centrifuged at >13,000 Â g for
5 min at 4
C for particle removal (see Notes 21 and 22).
4. Inject 10 μL of sample into the HPLC system (see Notes 23
and 24).
5. Run the binary-gradient elution profile: Initial conditions (75%
A; 25% B) increases linearly to 95% A in 10 min, then hold 95%
A for 7 min and raises to 100% A in 3 min, and is maintained
constant (100% A) for 3 min (see Note 25). Initial conditions
are restored in 5 min.
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Da ´ maso Hornero-Me ´ ndez
30
C. Alternatively, solvent can be evaporated under N 2
stream.
11. Dissolve the dry extract in 0.5–1.0 mL of acetone (see
Note 15) and stored at À30
C until HPLC analysis.
12. Extraction should be performed in at least triplicate.
3.4 Saponification
If saponification is required for the hydrolysis of lipids and carotenoid esters, the following micro-scale procedure can be used.
1. 1 mL of the pigment extract obtained in Subheading 3.2
(step 10) is placed in a 15 mL centrifuge tube (Falcon type
or similar) and dried under a nitrogen stream (see Note 19).
2. Add 2 mL of diethyl ether and 1 mL of 10% (w/v) KOH–
methanol.
3. Shake the mixture in a vortex for 30 s and left to react for
20 min at room temperature under nitrogen atmosphere and
with periodic shaking (see Note 20).
4. Add 8 mL of distilled water for washing, and separate phases by
centrifugation (at 4500 Â g at 4
C during 5 min).
5. Discard the lower aqueous phase and repeat the washing operation (see Note 13) until the pH of the aqueous phase is neutral
(use pH test strips).
6. Discard the lower phase (last washing).
7. Filter the ether phase through an anhydrous Na 2 SO 4 , and rinse
the filtering material with more diethyl ether. Collect the filtrate in a clean test tube.
8. Evaporate the ether to dryness under a nitrogen stream.
9. Dissolve the extract in 1 mL of acetone and store at À30
C
until subsequent chromatographic analysis.
3.5 High
Performance Liquid
Chromatography
(HPLC)
1. Equilibrate the HPLC equipment to the initial solvent conditions (75% solvent A and 25% solvent B) and 25
C for column
temperature.
2. Set flow rate at 1 mL/min.
3. Prior to injection, samples are centrifuged at >13,000 Â g for
5 min at 4
C for particle removal (see Notes 21 and 22).
4. Inject 10 μL of sample into the HPLC system (see Notes 23
and 24).
5. Run the binary-gradient elution profile: Initial conditions (75%
A; 25% B) increases linearly to 95% A in 10 min, then hold 95%
A for 7 min and raises to 100% A in 3 min, and is maintained
constant (100% A) for 3 min (see Note 25). Initial conditions
are restored in 5 min.
122
Da ´ maso Hornero-Me ´ ndez