10. Run the migration in EP/E 9:1 v/v solvent.
11. Plunge in primulin solution.
12. Observe at 365 nm. PDIM-A and PDIM-B are expected in
fractions 15–20, at Rf 0.75 and 0.65, respectively, and TAG is
expected in fractions 24–35, at Rf 0.35.
13. Pool fractions exhibiting similar lipid contents.
3.14 Purification of
MAME by
Preparative TLC
1. Dissolve MAME-enriched methanol precipitate (Subheading
3.9) in chloroform (to 10 mg/mL).
2. Spot this suspension as a line with a Pasteur pipette or a 100-μL
syringe on silica gel precoated plate, 20 cm  20 cm, 0.25 mm.
An equivalent of 7–10 mg per plate allows good separation.
3. Run the plate once in dichloromethane four or five times in
petroleum ether/diethyl ether 9:1 (v/v) for separation of ketoand methoxy-MA (Fig. 2a, b) [52, 53]. When several runs are
necessary, take care to stop the migration at the same level for
each run.
4. Visualize the spots by spraying the plate with 0.01% solution of
rhodamine B in 0.25 M sodium monophosphate, a reagent
that does not degrade the extracted lipid.
5. Scrap off the bands visualized by UV light.
6. Extract the lipid from the silica power with around 2 Â 3 mL of
diethyl ether.
7. Filter the ether extract, which should contain pure MAME, in a
test tube through a 1-cm Florisil Pasteur pipette, in order to
eliminate silica powder and rhodamine dye.
8. Evaporate the solvent under air stream.
3.15 Determination
of Number and
Configuration of
Double Bonds of
MAME by Argentation
Chromatography
To further separate purified MAME according to the presence, the
number, and the configuration of double bonds, argentation TLC
is used (see Note 17).
1. Prepare a solution of 10% (wt/vol) aqueous AgNO 3 .
2. Run precoated silica gel plates in the AgNO 3 solution. The
migration is slow around 3 h for running half of the plate.
3. Air-dry the impregnated TLC plate overnight at room
temperature.
4. Activate the plates at 100
C for 1 h prior to use.
5. Load the sample of purified MAME as described for preparative
TLC (see Subheading 3.14, step 7).
6. Run the TLC plate in dichloromethane.
7. Dry the plate at RT.
8. Spray with 0.2% dichlorofluorescein solution, a reagent that
does not degrade the extracted lipid.
132
Marie-Antoinette Lane ´ elle et al.
11. Plunge in primulin solution.
12. Observe at 365 nm. PDIM-A and PDIM-B are expected in
fractions 15–20, at Rf 0.75 and 0.65, respectively, and TAG is
expected in fractions 24–35, at Rf 0.35.
13. Pool fractions exhibiting similar lipid contents.
3.14 Purification of
MAME by
Preparative TLC
1. Dissolve MAME-enriched methanol precipitate (Subheading
3.9) in chloroform (to 10 mg/mL).
2. Spot this suspension as a line with a Pasteur pipette or a 100-μL
syringe on silica gel precoated plate, 20 cm  20 cm, 0.25 mm.
An equivalent of 7–10 mg per plate allows good separation.
3. Run the plate once in dichloromethane four or five times in
petroleum ether/diethyl ether 9:1 (v/v) for separation of ketoand methoxy-MA (Fig. 2a, b) [52, 53]. When several runs are
necessary, take care to stop the migration at the same level for
each run.
4. Visualize the spots by spraying the plate with 0.01% solution of
rhodamine B in 0.25 M sodium monophosphate, a reagent
that does not degrade the extracted lipid.
5. Scrap off the bands visualized by UV light.
6. Extract the lipid from the silica power with around 2 Â 3 mL of
diethyl ether.
7. Filter the ether extract, which should contain pure MAME, in a
test tube through a 1-cm Florisil Pasteur pipette, in order to
eliminate silica powder and rhodamine dye.
8. Evaporate the solvent under air stream.
3.15 Determination
of Number and
Configuration of
Double Bonds of
MAME by Argentation
Chromatography
To further separate purified MAME according to the presence, the
number, and the configuration of double bonds, argentation TLC
is used (see Note 17).
1. Prepare a solution of 10% (wt/vol) aqueous AgNO 3 .
2. Run precoated silica gel plates in the AgNO 3 solution. The
migration is slow around 3 h for running half of the plate.
3. Air-dry the impregnated TLC plate overnight at room
temperature.
4. Activate the plates at 100
C for 1 h prior to use.
5. Load the sample of purified MAME as described for preparative
TLC (see Subheading 3.14, step 7).
6. Run the TLC plate in dichloromethane.
7. Dry the plate at RT.
8. Spray with 0.2% dichlorofluorescein solution, a reagent that
does not degrade the extracted lipid.
132
Marie-Antoinette Lane ´ elle et al.