5. Collect and pool extracts into a separation funnel (1 L volume).
6. Add 50 mL of diethyl ether (containing 6 ppm BHT) and
100 mL of NaCl solution (10% w/v), and shake vigorously
during 1 min to facilitate carotenoid transfer to ether phase.
Allow to stand for the separation of two phases, and discard the
aqueous phase (lower phase).
7. The upper phase, containing the carotenoid pigments is
washed three additional times with 10% NaCl and finally with
2% (w/v) Na 2 SO 4 solution for removing the water contained
in the organic fraction (see Note 13). Aqueous phases are
discarded after each washing.
8. Filter the extract through an anhydrous Na 2 SO 4 bed into a
round-bottom flask. Wash the filtering material with additional
diethyl ether until no color is observed.
9. Evaporate the extract to dryness under vacuum in a rotary
evaporator at temperature not higher than 30
C (see Note 14).
10. The dry extract is dissolved in 3–5 mL of acetone (see Note 15)
and an aliquot (0.5 mL) is stored at À30
C until HPLC
analysis (see Note 16).
11. Extraction should be performed in at least triplicate (n ¼ 3).
3.3 Extraction
of Carotenoids
(Microscale
Procedure)
1. Sample size. Use 0.2–0.5 g depending on the sample nature
(see Note 17).
2. Place sample in a 15 mL centrifuge tube (Falcon tube or
similar) and extract carotenoids with 3 mL of acetone (containing 0.1% BHT) by using a homogenizer during 1 min (see
Notes 9 and 10). Add IS in this step (if used).
3. Separate extract by centrifugation at 4500 Â g at 4
C during
5 min.
4. Repeat the extraction procedure 3–4 times until complete
extraction of color.
5. Collect and pool extracts into a clean tube.
6. Reduce solvent volume down to 2–3 mL under a gentle nitrogen stream (see Note 18).
7. Add 3 mL of diethyl ether and 4 mL of NaCl solution (10%
w/v), and shake vigorously during 30 s to facilitate carotenoid
transfer to the ether phase.
8. Separate phases by centrifugation at 4500 Â g at 4
C during
5 min.
9. Transfer the upper phase (diethyl ether phase), with a glass
Pasteur pipette, into a clean tube, and repeat (2–3 times) the
extraction of the aqueous phase with 2 mL of diethyl ether.
Discard the lower phase (aqueous phase).
HPLC Analysis of Carotenoids
121
6. Add 50 mL of diethyl ether (containing 6 ppm BHT) and
100 mL of NaCl solution (10% w/v), and shake vigorously
during 1 min to facilitate carotenoid transfer to ether phase.
Allow to stand for the separation of two phases, and discard the
aqueous phase (lower phase).
7. The upper phase, containing the carotenoid pigments is
washed three additional times with 10% NaCl and finally with
2% (w/v) Na 2 SO 4 solution for removing the water contained
in the organic fraction (see Note 13). Aqueous phases are
discarded after each washing.
8. Filter the extract through an anhydrous Na 2 SO 4 bed into a
round-bottom flask. Wash the filtering material with additional
diethyl ether until no color is observed.
9. Evaporate the extract to dryness under vacuum in a rotary
evaporator at temperature not higher than 30
C (see Note 14).
10. The dry extract is dissolved in 3–5 mL of acetone (see Note 15)
and an aliquot (0.5 mL) is stored at À30
C until HPLC
analysis (see Note 16).
11. Extraction should be performed in at least triplicate (n ¼ 3).
3.3 Extraction
of Carotenoids
(Microscale
Procedure)
1. Sample size. Use 0.2–0.5 g depending on the sample nature
(see Note 17).
2. Place sample in a 15 mL centrifuge tube (Falcon tube or
similar) and extract carotenoids with 3 mL of acetone (containing 0.1% BHT) by using a homogenizer during 1 min (see
Notes 9 and 10). Add IS in this step (if used).
3. Separate extract by centrifugation at 4500 Â g at 4
C during
5 min.
4. Repeat the extraction procedure 3–4 times until complete
extraction of color.
5. Collect and pool extracts into a clean tube.
6. Reduce solvent volume down to 2–3 mL under a gentle nitrogen stream (see Note 18).
7. Add 3 mL of diethyl ether and 4 mL of NaCl solution (10%
w/v), and shake vigorously during 30 s to facilitate carotenoid
transfer to the ether phase.
8. Separate phases by centrifugation at 4500 Â g at 4
C during
5 min.
9. Transfer the upper phase (diethyl ether phase), with a glass
Pasteur pipette, into a clean tube, and repeat (2–3 times) the
extraction of the aqueous phase with 2 mL of diethyl ether.
Discard the lower phase (aqueous phase).
HPLC Analysis of Carotenoids
121