3.2 HPLC
Quantification of
Apocarotenoids
1. Weight 50 mg of lyophilized root material and put it in a 5 ml
glass vial (see Notes 8 and 9).
2. Add 50 μl of ribitol, used as internal standard to further yield
the polar fraction (see Note 10).
3. Extract with 400 μl of 80% MeOH.
4. Sonicate the mixture for 2 min at room temperature.
5. Centrifuge at 13,000 Â g for 5 min at room temperature and
collect the supernatant.
6. Repeat steps 3–5 two times more and put together the three
supernatants, obtaining a fraction of about 1.2 ml (see Note
11).
7. Take and aliquot of 300 μl of the supernatant and adjust it to a
final concentration of 0.5 M with KOH and incubate for 1 h at
room temperature (see Note 12).
8. Centrifuge at 13,000 Â g for 10 min and collect the supernatant. Samples are ready for their analysis by liquid chromatography (HPLC).
9. Perform HPLC with a system including a separation module.
Normally a system from Waters is used. The liquid chromatograph must be equipped with a 5 μm Nucleosil C 18 column
(250 Â 4 mm) or similar.
10. Use as solvent A: 1.5% H 3 PO 4 ; and solvent B: MeCN.
11. The sample injection volume is 20 μl.
12. For an optimal separation of the compounds use the following
gradient: a first step from 5% B to 25% B within 40 min, and
then from 25% B to 80% within 20 min.
13. Use a flow rate of ml min
À1 .
14. Detect the apocarotenoids photometrically at 250 and
380 nm, using a photodiode array detector (see Note 13).
15. Calculate quantitative values after standardization with ribitol
(used as internal standard), and using an appropriate software
(see Note 14).
4 Notes
1. We observed that with this ink, a better contrast was obtained.
However, other brands, such as Sheaffer or Pelikan can also
be used.
2. Washing tap water gives better results than with deionized
water. Moreover, the later can disrupt root membranes with
the corresponding loss of metabolites.
AM Symbiosis Quantification
297
Quantification of
Apocarotenoids
1. Weight 50 mg of lyophilized root material and put it in a 5 ml
glass vial (see Notes 8 and 9).
2. Add 50 μl of ribitol, used as internal standard to further yield
the polar fraction (see Note 10).
3. Extract with 400 μl of 80% MeOH.
4. Sonicate the mixture for 2 min at room temperature.
5. Centrifuge at 13,000 Â g for 5 min at room temperature and
collect the supernatant.
6. Repeat steps 3–5 two times more and put together the three
supernatants, obtaining a fraction of about 1.2 ml (see Note
11).
7. Take and aliquot of 300 μl of the supernatant and adjust it to a
final concentration of 0.5 M with KOH and incubate for 1 h at
room temperature (see Note 12).
8. Centrifuge at 13,000 Â g for 10 min and collect the supernatant. Samples are ready for their analysis by liquid chromatography (HPLC).
9. Perform HPLC with a system including a separation module.
Normally a system from Waters is used. The liquid chromatograph must be equipped with a 5 μm Nucleosil C 18 column
(250 Â 4 mm) or similar.
10. Use as solvent A: 1.5% H 3 PO 4 ; and solvent B: MeCN.
11. The sample injection volume is 20 μl.
12. For an optimal separation of the compounds use the following
gradient: a first step from 5% B to 25% B within 40 min, and
then from 25% B to 80% within 20 min.
13. Use a flow rate of ml min
À1 .
14. Detect the apocarotenoids photometrically at 250 and
380 nm, using a photodiode array detector (see Note 13).
15. Calculate quantitative values after standardization with ribitol
(used as internal standard), and using an appropriate software
(see Note 14).
4 Notes
1. We observed that with this ink, a better contrast was obtained.
However, other brands, such as Sheaffer or Pelikan can also
be used.
2. Washing tap water gives better results than with deionized
water. Moreover, the later can disrupt root membranes with
the corresponding loss of metabolites.
AM Symbiosis Quantification
297